Nutrition and Athletic Performance

Nutrition and
Athletic
Performance
Edited by
Stephen Ives
Printed Edition of the Special Issue Published in Nutrients
www.mdpi.com/journal/nutrients
Nutrition and Athletic Performance
Nutrition and Athletic Performance
Editor
Stephen Ives
MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin
Editor
Stephen Ives
Skidmore College
USA
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MDPI
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Performance).
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Contents
About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface to ”Nutrition and Athletic Performance” . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Diego Fernández-Lázaro, Juan Mielgo-Ayuso, Jesús Seco Calvo, Alfredo Córdova Martı́nez,
Alberto Caballero Garcı́a and Cesar I. Fernandez-Lazaro
Modulation of Exercise-Induced Muscle Damage, Inflammation, and Oxidative Markers by
Curcumin Supplementation in a Physically Active Population: A Systematic Review
Reprinted from: Nutrients 2020, 12, 501, doi:10.3390/nu12020501 . . . . . . . . . . . . . . . . . . . 1
Mojtaba Kaviani, Philip D. Chilibeck, Spencer Gall, Jennifer Jochim and Gordon A. Zello
The Effects of Low- and High-Glycemic Index Sport Nutrition Bars on Metabolism and
Performance in Recreational Soccer Players
Reprinted from: Nutrients 2020, 12, 982, doi:10.3390/nu12040982 . . . . . . . . . . . . . . . . . . . 21
Kengo Ishihara, Natsuki Uchiyama, Shino Kizaki, Emi Mori, Tsutomu Nonaka and Hiroshi
Oneda
Application of Continuous Glucose Monitoring for Assessment of Individual Carbohydrate
Requirement during Ultramarathon Race
Reprinted from: Nutrients 2020, 12, 1121, doi:10.3390/nu12041121 . . . . . . . . . . . . . . . . . . 35
Jorge Pérez-Gómez, Santos Villafaina, José Carmelo Adsuar, Eugenio Merellano-Navarro

and Daniel Collado-Mateo
Effects of Ashwagandha (Withania somnifera) on VO2max : A Systematic Review
and Meta-Analysis
Csilla Ari, Cem Murdun, Craig Goldhagen, Andrew P. Koutnik, Sahil R. Bharwani, David
M. Diamond, Mark Kindy, Dominic P. D’Agostino and Zsolt Kovacs
Exogenous Ketone Supplements Improved Motor Performance in Preclinical Rodent Models
Álvaro Huerta Ojeda, Camila Tapia Cerda, Marı́a Fernanda Poblete Salvatierra, Guillermo
Barahona-Fuentes and Carlos Jorquera Aguilera
Effects of Beta-Alanine Supplementation on Physical Performance in Aerobic–Anaerobic
Transition Zones: A Systematic Review and Meta-Analysis
Carlos Rodrigo Soares Freitas Sampaio, Felipe J. Aidar, Alexandre R. P. Ferreira, Jymmys
Lopes dos Santos, Anderson Carlos Marçal, Dihogo Gama de Matos, Raphael Fabrı́cio
de Souza, Osvaldo Costa Moreira, Ialuska Guerra, José Fernandes Filho, Lucas Soares
Marcucci-Barbosa, Albená Nunes-Silva, Paulo Francisco de Almeida-Neto, Breno Guilherme
Araújo Tinoco Cabral and Victor Machado Reis
Can Creatine Supplementation Interfere with Muscle Strength and Fatigue in Brazilian National
Level Paralympic Powerlifting?
Tomáš Hlinský, Michal Kumstát and Petr Vajda

Effects of Dietary Nitrates on Time Trial Performance in Athletes with Different Training Status:
Systematic Review
v
Pavel Kysel, Denisa Haluzı́ková, Radka Petráková Doležalová, Ivana Laňková, Zdeňka
Lacinová, Barbora Judita Kasperová, Jaroslava Trnovská, Viktorie Hrádková, Miloš Mráz,
Zdeněk Vilikus and Martin Haluzı́k
The Influence of Cyclical Ketogenic Reduction Diet vs. Nutritionally Balanced Reduction Diet
on Body Composition, Strength, and Endurance Performance in Healthy Young Males: A
Randomized Controlled Trial
Néstor Vicente-Salar, Guillermo Santos-Sánchez and Enrique Roche

Nutritional Ergogenic Aids in Racquet Sports: A Systematic Review
Monique D. Dudar, Emilie D. Bode, Karly R. Fishkin, Rochelle A. Brown, Madeleine M.

Carre, Noa R. Mills, Michael J. Ormsbee and Stephen J. Ives
Pre-Sleep Low Glycemic Index Modified Starch Does Not Improve Next-Morning Fuel Selection
or Running Performance in Male and Female Endurance Athletes
Maija Marttinen, Reeta Ala-Jaakkola, Arja Laitila and Markus J. Lehtinen

Gut Microbiota, Probiotics and Physical Performance in Athletes and Physically
Active Individuals
Yasuko Yoshida, Keisei Kosaki, Takehito Sugasawa, Masahiro Matsui, Masaki Yoshioka, Kai
Aoki, Tomoaki Kuji, Risuke Mizuno, Makoto Kuro-o, Kunihiro Yamagata, Seiji Maeda and
Kazuhiro Takekoshi
High Salt Diet Impacts the Risk of Sarcopenia Associated with Reduction of Skeletal Muscle
Performance in the Japanese Population
vi
About the Editor
Stephen Ives
Dr. Stephen Ives, Associate Professor and Associate Chair, Health and Human Physiological
Sciences, Skidmore College. I approach the understanding of health and human physiology with
an integrative physiological perspective, and thus I am interested in how lifestyle factors such as
nutrition influence our health but also our physical capacity for exercise, with a desire to understand
these phenomena in a systemic way.
vii
Preface to ”Nutrition and Athletic Performance”
The current collection of articles selected for this Special Issue, and now monograph, aimed to
increase our understanding of the role of nutrition in athletic performance. The aim was to gather
new evidence, or novel syntheses of existing evidence, in various models that address how diet,
dietary supplementation, or manipulating the temporal nature of diet/supplementation may alter or
influence human performance positively or negatively across broad populations. Indeed, I believe we
were successful, as the current collection of papers includes such timely topics as the ketogenic diet
or ketone supplementation, the gut microbiome, understanding the role of the glycemic index and
timing, as well as supplementation with nitrates, curcumin, beta alanine, and creatine. Some of these
papers address the current state of affairs regarding existing practices, which is frequently a forgotten
or overlooked aspect that is often crucial before considering dietary manipulation. Importantly, this
collection of studies was inclusive of age, sex/gender, ability status, and diversity of sport or activity.
I would like to personally thank the authors for contributing to this Special Issue, which will now be
memorialized as a monograph, but also the reviewers who provided critical feedback in improving
the manuscripts presented herein, but also in reviewing articles that were not deemed acceptable
in the journal and Special Issue. I hope that this collection helps the researchers, practitioners, and
students who will become the next generation that will continue such academic pursuits.
Stephen Ives
Editor
ix
nutrients
Review
Modulation of Exercise-Induced Muscle Damage,
Inflammation, and Oxidative Markers by Curcumin
Supplementation in a Physically Active Population:
A Systematic Review
Diego Fernández-Lázaro 1, *, Juan Mielgo-Ayuso 2 , Jesús Seco Calvo 3 ,
Alfredo Córdova Martínez 2 , Alberto Caballero García 4 and Cesar I. Fernandez-Lazaro 1,5
1 Department of Cellular Biology, Histology and Pharmacology, Faculty of Health Sciences, University of
Valladolid, Campus of Soria, 42003 Soria, Spain; [email protected]
2 Department of Biochemistry and Physiology, Faculty of Health Sciences, University of Valladolid, Campus
of Soria, 42003 Soria, Spain; [email protected] (J.M.-A.); [email protected] (A.C.M.)
3 Institute of Biomedicine (IBIOMED), Physiotherapy Department, University of Leon, Campus of Vegazana,
24071 Leon, Spain; [email protected]
4 Department of Anatomy and Radiology, Faculty of Health Sciences, University of Valladolid, Campus of
Soria, 42003 Soria, Spain; [email protected]
5 Department of Preventive Medicine and Public Health, School of Medicine, University of Navarra, IdiSNA,
31008 Pamplona, Spain
* Correspondence: [email protected]; Tel.: +34-975-129-185
Received: 22 January 2020; Accepted: 12 February 2020; Published: 15 February 2020
Abstract: Physical activity, particularly high-intensity eccentric muscle contractions, produces

exercise-induced muscle damage (EIMD). The breakdown of muscle fibers and the consequent
inflammatory responses derived from EIMD affect exercise performance. Curcumin, a natural
polyphenol extracted from turmeric, has been shown to have mainly antioxidant and also
anti-inflammatory properties. This effect of curcumin could improve EIMD and exercise performance.
The main objective of this systematic review was to critically evaluate the effectiveness of curcumin
supplementation on EIMD and inflammatory and oxidative markers in a physically active population.
A structured search was carried out following Preferred Reporting Items for Systematic Review and
Meta-Analyses (PRISMA) guidelines in the databases SCOPUS, Web of Science (WOS), and Medline
(PubMed) from inception to October 2019. The search included original articles with randomized
controlled crossover or parallel design in which the intake of curcumin administered before and/or
after exercise was compared with an identical placebo situation. No filters were applied to the type of
physical exercise performed, the sex or the age of the participants. Of the 301 articles identified in the
search, 11 met the established criteria and were included in this systematic review. The methodological
quality of the studies was assessed using the McMaster Critical Review Form. The use of curcumin
reduces the subjective perception of the intensity of muscle pain; reduces muscle damage through the
decrease of creatine kinase (CK); increases muscle performance; has an anti-inflammatory effect by
modulating the pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-8; and may have a slight
antioxidant effect. In summary, the administration of curcumin at a dose between 150–1500 mg/day
before and during exercise, and up until 72 h’ post-exercise, improved performance by reducing
EIMD and modulating the inflammation caused by physical activity. In addition, humans appear to
be able to tolerate high doses of curcumin without significant side-effects.
Keywords: natural polyphenols; curcumin; muscle-damaging exercise; anti-inflammatory;

antioxidants; physical activity
Nutrients 2020, 12, 501; doi:10.3390/nu12020501 www.mdpi.com/journal/nutrients
1
Nutrients 2020, 12, 501
1. Introduction
Physical activity, particularly high-intensity eccentric muscle contractions, induces
exercise-induced muscle damage (EIMD) [1,2]. EIMD leads to the onset of an inflammatory response
that is associated with a decrease in the ability to generate muscle strength, decreased range of
motion (ROM), localized swelling, delayed onset muscle soreness (DOMS), and increased muscle
proteins in the blood (creatine kinase (CK), lactate dehydrogenase (LDH), and myoglobin (Mb)) [3]. In
addition, EIMD triggers inflammatory responses that result in elevations of inflammation markers
such as C-reactive protein (CRP) and some inflammatory interleukins (IL-1, IL-6, tumor necrosis factor
(TNF-α)) [4]. Similarly, it promotes the production of transcription factors such as nuclear factor kB
(NF kB) through the production of reactive oxygen species (ROS) [5].
On the other hand, research indicates that oxidative stress (OS) is evident following EIMD by
an increase in ROS [6]. In this sense, endogenous antioxidants may also be up-regulated via exercise,
which stimulates an acute OS and inflammatory response [7]. Therefore, inflammatory processes are
always linked to OS and must be analyzed and controlled together, because both are directly involved
in EIMD [8]. One way to prevent and minimize the effects of OS and the inflammatory process, and
attenuate EIMD [9], could be an oral intake of anti-inflammatory or antioxidant supplementation. A
natural product that can be used, with potential antioxidant and anti-inflammatory effects, is curcumin
(1,7-bis (4-hydroxy-3-methoxyphenyl) 1,6-heptadiene-3,5-dione), which is the main natural bioactive
polyphenol of the spice herb turmeric (2%–5% by weight). Curcumin is a highly pleiotropic molecule
that interacts with multiple anti-inflammatory and antioxidant pathways [10,11]. The United States
Food and Drug Administration (FDA) has listed curcumin as GRAS (generally recognized as safe), and
curcumin-containing supplements have been approved for human ingestion [12]. In this way, curcumin
used as a pharmaceutical preparation has been shown to be safe, even at high doses. However, it
has been shown to cause some gastric irritation in humans, hepatotoxicity in mice, and at high doses,
hepatotoxicity in rats. Humans appear to be able to tolerate high doses of curcumin without significant
side-effects. This may be because of differences in metabolism of curcumin in humans as compared to
susceptible species such as rats. However, when used as a spice, because of its high water content, it
could be attacked by aflatoxin-producing fungi, causing kidney, lung, or liver toxicity. It must be taken
into account that curcumin as a spice is produced in tropical countries (warm and humid) that favor
the growth of fungi [11,12].
Curcumin supplementation could be beneficial for attenuating EIMD given that curcumin has been
shown to potentially help alleviate exercise performance decrements following intense and challenging
exercise, as a result of membrane protective effects, antioxidants response, and anti-inflammatory
action [13,14]. The anti-inflammatory properties attributed to curcumin are due to its ability to inhibit
the nuclear factor kappa (NF-κB), which may be a muscle protective and regeneration agent and plays
an important role in controlling physiological mechanisms of inflammation and protein breakdown [15].
Curcumin is capable of blocking the activation of TNF-α-dependent NF-κB and the activation pathway
induced by ROS [16–18]. Likewise, curcumin could have a low regulatory effect on the expression
of the COX-2 enzyme and inhibit pro-inflammatory enzyme 5-LOX (lipoxygenase-5) expression in
the leukotriene-producing metabolic pathway [19], as well as the intercellular adhesion molecule
1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)—a crucial step in the inflammatory
response—and decrease inducible nitric oxide synthase (iNOS), which is directly responsible for
inflammatory damage by blocking of the cytokines responsible for its activation [19,20]. In this
way, curcumin induces the negative regulation of pro-inflammatory interleukins (IL-1, IL-2, IL-6,
IL-8, and IL-12), inflammatory cytokines, such as TNF-α and monocyte-1 chemotherapeutic protein
(MCP-1), through inhibition of the transcription signaling pathway (JAK/STAT) [11]. In addition, the
overexpression of Bcl-2 or Bcl-X L protects cells from apoptosis that counteracts proapoptotic and
proinflammatory attacks and restores the anti-inflammatory physiological phenotype [18,21]; curcumin
controls the response to thermal shock for attenuated muscle damage [22] and biomarkers of muscle
damage, such as CK [11].
2
Nutrients 2020, 12, 501
In line with this, some studies have shown the effects of curcumin supplementation on OS,
inflammation, EIMD, and sport performance with controversial results. These inconsistent results
may be due, in part, to differences in doses, timing of supplementation, timing of exercise, exercise
model, and experimental design between studies [23]. Previous research has indicated that curcumin
may have antioxidant, anti-inflammatory, and analgesic effects on DOMS [24]. Furthermore, similar
effects of curcumin have been described by Tanabe et al. [1], who found that ingestion before exercise
could attenuate acute inflammation, and after exercise, it could attenuate muscle damage and facilitate
faster recovery. Drobnic et al. [5] reported a reduction in muscular trauma with a moderate reduction
in pain with curcumin supplementation. In contrast, Sciberras et al. [4] did not reveal any statistical
difference between intervention with curcumin and placebo in levels of IL-6 and IL-10. In addition,
markers of oxidative stress were only slightly increased after exercise in both groups, which does not
allow a comparison of the effects of curcumin versus placebo [5], and there were no differences in
terms of changes in maximal voluntary contraction (MVC) and serum CK activity [25]. Finally, not all
observed changes in performance and soreness after exercise in humans [10] have been reproduced on
the mouse model [26].
However, it is necessary to clarify the useful doses, timing (before or after exercise), duration of
treatment, and the effects of curcumin on OS, inflammation, and EIMD. Therefore, the purpose of this
study was to critically evaluate the effectiveness of curcumin supplementation on EIMD, inflammatory,
and oxidative markers in a physically active population. In addition, the study shows the effective
doses, timing, and duration of treatment for optimal application.
2. Material and Methods
2.1. Search Strategy

The present article is a systematic review focusing on the effect of curcumin supplementation
on muscle pain, muscle function, muscle enzyme activity, inflammatory markers, and antioxidant
effect and was conducted following the Preferred Reporting Elements for Systematic Reviews and
Meta-analysis (PRISMA) guidelines [27] and the PICOS question model for the definition of inclusion
criteria: P (population); “healthy exercise practitioners”, I (intervention); “supplementation with
curcumin”, C (comparison); “same conditions with placebo or control group”, O (outcomes); “muscle
pain, inflammation and/or muscle damage serum markers and antioxidant effect”, S (study design);
“double- or single-blind design and randomized parallel or crossed”.
A structured search was carried out in the databases SCOPUS, Medline (PubMed), and Web of
Science (WOS), which includes other databases such as BCI, BIOSIS, CCC, DIIDW, INSPEC, KJD,
MEDLINE, RSCI, SCIELO, all of which are high-quality databases which guarantee good bibliographic
support. The search covered a time span from March 2015—when Nicol et al. [28] suggested the
use of oral curcumin is likely to reduce pain associated with delayed onset muscle soreness (DOMS)
with some evidence for enhanced recovery of muscle performance—to November 2019. Search
terms were a mix of Medical Subject Headings (MeSH) and free words for key concepts related to
curcumin, muscle, exercise, inflammation, and recovery, as follows: (“curcumin” OR “curcuminoids”
OR “curcuma longa” OR “turmeric”) AND (“muscle damage” OR “delay onset muscle soreness”
OR “DOMS” OR “inflammation” OR “inflammatory” OR “inflammatory markers” OR “oxidative
stress”) AND (“exercise” OR “physical activity” OR “sports”). Through this search, relevant articles in
the field were obtained applying the snowball strategy. All titles and abstracts from the search were
cross-referenced to identify duplicates and any potential missing studies. Titles and abstracts were
then screened for a subsequent full-text review. The search for published studies was independently
performed by two authors (DFL and CIFL) and disagreements about physical parameters were resolved
through discussion.
3
Nutrients 2020, 12, 501
2.2. Selection of Articles: Inclusion and Exclusion Criteria

For the articles obtained in the search, the following inclusion criteria were applied to select studies:
articles (I) depicting a well-designed experiment that included the ingestion of a dose of curcumin,
or a curcumin-containing product, before and/or during exercise in humans; (II) with an identical
experimental situation with or without the ingestion of a placebo; (III) with a double- or single-blind
design and randomized parallel or crossed design; (V) with clear information on the administration
of curcumin (dose of curcumin per kg body mass and/or absolute dose of curcumin on body mass,
time of curcumin intake before or after exercise, duration of treatment); (VI) in which curcumin was
administered in the form of a beverage, gum, or pills; (VII) in which one of the measured variables was
changes in muscle pain, muscle function, muscle enzyme activity, inflammatory markers, or antioxidant
effect; (VIII) in which the languages were restricted to English, German, French, Italian, Spanish, and
Portuguese. The following exclusion criteria were applied: (I) animal studies, (II) uncontrolled trials,
(III) studies using non-standardized turmeric extracts or extracts of unknown curcuminoid content,
and (IV) studies performed on subjects with a prior condition of musculoskeletal injury or pain. There
were no filters applied to the individuals’ fitness level, sex, or age to increase the power of the analysis.
The methodological quality of the articles, evaluated using McMaster’s Critical Review Form [29],
scored between 12 and 15 points, representing a minimum methodological quality of 75% and a
maximum of 93.8%. Of the 11 studies, 7 achieved a “Very Good” quality, 2 a “Good” quality, and 2
studies an “excellent” quality. No study was excluded because it did not reach the minimum quality
threshold. Table 1 details the results of the criteria evaluated, where the main deficiencies found
in methodological quality are associated with items 5 and 12 of the questionnaire, and comprises a
detailed justification of the size of the study and a discussion of relevance of the results to clinical
practice, respectively. The objective of this evaluation was to determine the existing methodological
limitations in each of the studies and to allow the quality of the results to be comparable between the
different study designs.
Once the inclusion/exclusion criteria were applied to each study including authors, year of
publication, study design, curcumin administration (dose and timing), sample size, and characteristics
of the participants (fitness level and sex), and final outcomes of the interventions were extracted
independently by two authors (DFL and CIFL) using a spreadsheet (Microsoft Inc, Seattle, WA, USA).
Subsequently, disagreements were resolved through discussion until a consensus was reached.
4
Table 1. Methodological quality of the studies included in the systematic review.
Nakhostin-
Drobnic Sciberras Nicol et Tanabe McFarlin Delecroix Tanabe Tanabe Jäger et Basham
Roohi et
References et al., et al., al., 2015 et al., et al., et al., et al., et al., al., 2019 et al., TI
al., 2016
2014 [5] 2015 [4] [28] 2015 [2] 2016 [30] 2017 [31] 2018 [1] 2019 [25] [10] 2019 [32]
[24]
Nutrients 2020, 12, 501
1 1 1 1 1 1 1 1 1 1 1 1 10
2 1 1 1 1 1 1 1 1 1 1 1 10
3 1 1 1 1 1 1 1 1 1 1 1 10
4 1 1 1 1 1 1 1 1 1 1 1 10
5 0 1 0 1 0 0 0 1 1 1 1 4
6 0 1 0 1 1 1 1 1 1 1 1 7
7 1 1 1 1 1 1 1 1 1 1 1 10
8 1 1 1 1 1 1 1 1 1 1 1 10
ITEMS
9 1 1 1 1 1 1 1 1 1 1 1 10
10 1 1 0 1 1 1 0 1 1 1 1 7
11 1 1 1 1 1 1 1 1 1 1 1 10
12 1 0 0 1 1 0 0 0 0 0 0 3
13 0 0 1 0 1 1 1 1 1 1 1 6
5
14 1 1 1 1 1 1 1 1 1 1 1 10
15 1 1 1 1 1 1 0 1 0 1 0 7
16 1 1 1 0 0 0 1 1 1 1 1 6
TS 13 14 12 14 14 13 12 15 14 15 14
% 81.3 87.5 75 87.5 87.5 81.3 75 93.8 87.5 93.8 87.5
MQ VG VG G VG VG VG G E VG E VG
(TS ) Total items fulfilled by study. (1) Criterion met; (0) Criterion not met. (TI ): Total items fulfilled by items. Methodological Quality (MQ): poor (P) ≤8 points; acceptable (A) 9–10 points;
good (G) 11–12 points; very good (VG) 13–14 points; excellent (E) ≥15 points.
Nutrients 2020, 12, 501
3. Results
3.1. Selection of Studies

The literature search provided a total of 301 articles related to the select descriptors, but only
11 articles met all the inclusion/exclusion criteria (Figure 1). The number of the articles to which
each exclusion criterion was applied were as follows: 94 papers were removed because they were
duplicates. After the elimination of duplicate articles, 207 articles were selected for examination by
title and abstract, of which 150 were excluded as non-intervention studies and 38 as unrelated to the
search topic. The full texts of the remaining 19 publications were evaluated according to the inclusion
criteria, from which three studies were eliminated because they were conducted in animal populations,
four because they used unhealthy subjects, and one because they did not measure any of the variables
included in this study.
Figure 1. Selection of Studies.
3.2. Characteristics of the Studies

The participants ‘samples (n = 237) included individuals of both genders (187 men and 50 women),
where 10 were elite athletes, 131 were moderately active people, and 96 were people who were
asked not to undergo pre-study training (Table 2). In ten of the eleven articles, some commercial
supplement of curcumin in capsules of standardized composition and known bioavailability was used,
while Nicol et al. [28] opted for a pharmaceutical preparation of curcumin capsules using a specific
composition protocol for research. In addition, Delecroix et al. [31] chose a combination of curcumin
plus piperine in the composition. Regarding the daily dose of curcumin, seven studies used doses
ranging from 150 to 1500 mg [1,2,4,5,10,24,25,30,32], and two studies tested higher doses of about
5 g [28] and 6 g [31] daily. In nine of the included studies, supplementation was given before and after
exercise [1,2,5,10,25,28,30–32], Sciberras et al. [4] used curcumin before exercise, and Nakhostin-Roohi
et al. [24] supplemented with curcumin after exercise. Finally, treatment duration ranged from one to
fifty-six days, with three studies of four days [1,5,31], two of six days [28,30], two of seven days [1,25],
two of one day [2,24], one of twenty-eight days [10], and one of fifty-six days [32], respectively.
6
Nutrients 2020, 12, 501
Table 2. Characteristics of participants and interventions in the studies included in the review.
Elite Athletes 1 Study [31]

Level of Participants
Moderately Active 5 Studies [4,5,10,28,32]
No Regular Training before the
5 Studies [1,2,24,25,30]
Study
Commercially available curcumin
Type of Administration of 10 studies [1,2,4,5,10,24,25,30–32]
supplement
Curcumin
Curcumin capsule made for the
1 study [28]
study
150 mg/day 1 study [24]
180 mg/day (2 doses of 90 mg/day) 2 studies [1,25]
300 mg /day (2 doses of 150
1 study [2]
mg/day)
Dosage Used
400 mg/day 1 study [30]
400 mg /day (2 doses of 200
1 study [5]
mg/day)
500 mg / day 1 study [4]
5 g/day (2 doses of 2.5 g/day) 1 study [28]
6 g of curcumin + 60 mg of
piperine/day (3 doses of 2 g of 1 study [31]
curcumin + 20 mg of piperine/day)
600 mg/day (3 doses of 200 mg of
1 study [10]
curcumin)
1500 mg/day (3 doses of 500 mg of
1 study [32]
curcumin)
Before Exercise 1 study [4]
Moment of Supplementation
Before and After Exercise 9 studies [1,2,5,10,25,28,30–32]
After Exercise 1 study [24]
1 day 2 studies [2,24]
4 days 3 studies [4,5,31]
Duration of Treatment 6 days 2 studies [28,30]
7 days 2 studies [1,25]
56 days 1 study [10]
28 days 1 study [32]
3.3. Outcome Measures

Table 3A–C includes information about the author/s and year of publication; the sample
investigated, with details of fitness level, sex, and the number of participants; the study design
cites the control group if the study included one; the supplementation protocol that specifies the type
of curcumin used, the dose, and the time at which it was administered; the parameters analyzed or
main effects on muscle damage; and finally, results or main conclusions.
7
Table 3. Summary of studies included in this systematic review.
A. Summary of Studies Included in This Systematic Review.

Author/s—Year Study Design Population Intervention Analyzed Results Main Conclusions
200 mg curcumin Evidence of muscle

↓ RT and LT posterior and medial
Randomized 20 moderately active capsules (Phytosome injury by MRI
Drobnic et al., 2014 [5] controlled trial men (38.1 ± 11.1 years Meriva) twice a day CRP, hsCRP, ERS,
Nutrients 2020, 12, 501
single-blind and 32.7 ± 12.3 years) 48 h before exercise MCP-1, FRAP, CAT, † CRP, hsCRP, ERS, MCP-1, FRAP, CAT, GPx, CK
and for 24 h after GPx, CK
IL-8 ↓ IL-8
Intensity of pain † Intensity of pain
Double-blind 500 mg of curcumin RPE † RPE
randomized 11 male recreational in capsules (Meriva
Sciberras et al., 2015 Cortisol, PCR, Hto,
cross-over. Subjects athletes (35.5 ± 5.7 Curcumin) 72 h † Cortisol, PCR, Hto, Hb,
[4] Hb,
performed three trials years) before and
in total immediately before WBC, Neutrophils,
WBC, Neutrophils, IL-6,
(supplement/placebo exercise IL-6,
and control)
IL1-RA, IL-10 IL1-RA, IL-10
Questionnaire
↑ “better than usual”
DALDA
↓ Muscle pain: squatting jump (1.5−1.1; ± 1.2); Stretch butt
8
Muscle pain—VAS (−1.0a-1.9; ± 0.9); Sitting on one leg (−1.4a−1.7; 90% CL ±
Double-blind 2.5 g of curcumin in
1.0)
crossover 17 moderately active capsules, 48 h before
Nicol et al., 2015 [28]
randomized men (33.8 ± 5.4 years) the exercise and for Jump height to one
↑ Jump 1 leg (15%; 90% CL ± 1 2%)
controlled trial 72 h after leg
↓ CK 24 and 48 h before (−22%; 90% CL ± 22%), (−29%; ±
CK
21%)
↑ IL-6 at 0-h (31%; ± 29%) and 48 h (32%; ± 29%) ↓ 24 h
IL-6
post-exercise (−20%; ± 18%)
TNF-α † TNF-α
MVC Torque ↓ MVC Torque
150 mg curcumin
Single-blind 14 young men capsules ROM † ROM
crossover without regular (Theracurcumin Upper arm
Tanabe et al., 2015 [2] † Upper arm circumference
randomized resistance training Theravalues) twice a circumference
controlled trial (23.5 ± 2.3 years) day 1 h before
exercise and 12 h later Muscle pain—VAS †VAS
CK ↓ CK (maximum activity)
IL-6 † IL-6
TNF-α † TNF-α
B. Summary of Studies Included in This Systematic Review (Continued)
Table 3. Cont.
Subjective quadriceps
† Subjective quadriceps pain
pain
28 men and women without 400mg curcumin capsules ADL †ADL
Randomized
McFarlin et al., 2016 (Long-life)
controlled trial regular resistance training CK ↓CK
[30] 48h before exercise and for 72h
double blind (20 ± 1 ages and 19 ± 2 ages)
after TNF-α ↓TNF-α
Nutrients 2020, 12, 501
IL-6 †IL-6
IL-8 ↓IL-8
IL-10 †IL-10
Muscle pain—VAS ↓ VAS 48–72 h
Controlled test 10 young men without 150 mg of curcumin gin
Nakhostin-Roohi et TAC ↑ TAC
randomized crossed regular training with weights capsules (Theravalues)
al., 2016 [24] CK ↓ CK
double-blind (25.0 ± 1.6 years) Immediately after exercise
ALT ↓ ALT
AST ↓ AST
< Group reduction EXP: (−1.77 ± 7.25%; 1277 ±
2 g curcumin + 20 mg of 6-s power sprint 153 W). CON Group (−13.6 ± 13.0%; 1130 ± 241
piperine in capsules (MGD W)
Delecroix et al., 2017 A randomized, 10 rugby players elite level
Nature) 3 times/day48h before CMJ ↑ CMJ (ES = −0.56; CI 90% = 0.81−0.32)
[31] balanced cross-over (20.7 ± 1.4 years)
9
exercise and for 48 h after
exercise CK † CK 24, 48, 72 h post-exercise
Muscle
† Muscle pain—Hooper scale
pain—Hooper scale
Subjective quadriceps
† Subjective quadriceps pain
pain
MVC Torque Exp1:† Exp2:↑ MVC Torque
ROM Exp1:† Exp2:↑ ROM
Exp1: 10 men (28.5 ± 3.4 90mg curcumin capsules
Double-blind Muscle pain -VAS Exp1:† Exp2: ↓ VAS
years) Exp2: 10 men (29.0 ± (Theracurcumin Theravalues) 2
crossover
Tanabe et al., 2018 [1] times/day Exp1: 7 days before T2 Exp1:† Exp2: † T2
randomized 3.9 years) Both untrained 3-7
days prior to assay exercise Exp2: 7 days after
controlled trial CK Exp1:† Exp2:↓ CK
exercise
TNF- α Exp1:† Exp2: † TNF- α
IL-8 Exp1: ↓ Exp2: † IL-8
d-ROMs Exp1:† Exp2: † d-ROMs
BAP Exp1:† Exp2: † BAP
24 young men without 90 mg curcumin capsules MVC Torque PRE: † POST: † MVC Torque
Tanabe et al., 2019 Single-Blind Parallel intense training during the (Theracurcumin Theravalues)
ROM PRE: † POST: ↑ ROM
[25] Randomized Trial study period PRE (28.8 ± 3.6 twice a day PRE: 7 days before
years) POST (29.8 ± 3.4 years) exercise POST: 4 days after Muscle pain—VAS PRE: † POST: ↓ VAS
CON (28.0 ± 3.2 years) exercise CON: 4 days after
CK PRE: † POST: † CK
exercise
Table 3. Cont.
C. Summary of Studies Included in This Systematic Review (Continued)

Subjective muscle
pain anterior,
G1: Placebo (PLB) G2:
posterior, and total
50 mg curcumin in
Nutrients 2020, 12, 501
scale 100 mm
63 men (31) and capsules
Maximum extension
Randomized women (32) (21 ± 2 (CurcuWIN® )G3: 200 ↑ Subjective muscle pain (anterior, posterior) G 1, 2, and 3 †
torque and isokinetic
Jäger et al., 2019 [10] controlled trial years) physically mg curcumin in Subjective (total) muscle pain G3 1 h and 24 h post-exercise
flexion Extension
double-blind active meeting ACSM capsules † Maximum bending torque G2 † Bending power G2
® power and isokinetic
guidelines (CurcuWIN )3
flexion Isometric
times/day
torque Measurements:
(breakfast/lunch/dinner)
1 h, 24 h, 48 h, and 72
h post-exercise
1.5 g curcumin/69 mg
20 men elite level
curcuminoids 500 mg Oxidative stress
Randomized (21.7 ± 2.9 years)
Basham et al., 2019 capsule (CurcuFresh, Inflammation
controlled trial physically active ↓ CK (p < 0.0001) ↓ VAS (p = 0.012) &TAC &MDA &TNF-α
[32] NOW FoodsUSA) Muscle damage
double-blind compliance with
twice a day (2 Muscle pain
ACSM guidelines
breakfast/1 dinner)
(A) ↑: Statistically significant increase; †: change without statistical significance; ↓: Statistically significant decrease; MRI: magnetic resonance imaging; RT: right thigh; LT: left thigh; CRP:
10
C-reactive protein; hsCRP: high sensitivity CRP; ERS: erythrocyte sedimentation; MCP-1: monocyte 1 chemotherapeutic protein; FRAP: Ferric reduction capacity of plasma; CAT:
catalase; GPx: glutathione peroxidase; CK: creatine kinase; IL-8: interleukin 8; RPE: subjective perception of effort; Hto: hematocrit; Hb: hemoglobin; WBC: white blood cell count; IL-6:
interleukin 6; IL1-RA: interleukin 1-RA; IL-10: interleukin 10; DALDA: daily analysis of life demands in athletes; VAS: visual analog scale; TNF-α: tumor necrosis factor alpha; CVS:
maximum voluntary contraction; ROM: range of motion; TAC: Total Antioxidant Capacity; ALT: alanine aminotransferase; AST: aspartate aminotransferase. (B) ADL: Activities of
daily living; CMJ: Contra movement jump; MVC Torque: Maximum voluntary contraction torque; ROM: Range of motion; T2 : Transverse relaxation time; d-ROMs: Derivatives of
reactive oxygen metabolites; BAP: Biological antioxidant potential; EXP: Experimental; CON: Control. (C) &: Unchanged; PLB: Placebo; G: Group; MDA: Malondialdehyde; ACSM: The
American College of Sports Medicine.
Nutrients 2020, 12, 501
4. Discussion
The main objective of this systematic review was to critically evaluate the effectiveness of
curcumin supplementation on EIMD (muscle pain, muscle performance, and muscle enzyme activity)
and inflammatory and oxidative markers in a physically active population. The main results indicated
that supplementation with 150 and 1500 mg/day of oral curcumin, both before and up until 72 h after
exercise, has been shown to be effective on exercise performance, modulated in part by the reduction
of EIMD and inflammation caused by physical activity. However, it was difficult to determine the
true efficacy of the antioxidant capacity of curcumin. Due to the differently measured outcomes in
the studies, the following outcomes were divided into different groups to provide a clearer analysis.
The results could be influenced by type of exercise, amount of each supplement, and duration of the
intervention. Participant characteristics, such as age, gender, ethnicity, body composition, training
level, differences in training, nutrition, and health status, may also have influenced the results.
4.1. Curcumin Supplementation

The dose of curcumin administered in interventions ranged from 150 to 6000 mg/day, and
therefore, the effects of curcumin in a physically active population should be attributed to this dose
range. However, the European Food Safety Authority (EFSA) determined the permitted daily intake to
be 3 mg/kg body weight [33]. Thus, two investigations justified a dose of 180 mg/day of curcumin
following the EFSA recommendation [1,25]. However, other studies [28,30,32] did not base the choice
of dose on the same criteria.
Mc Farlin et al. [30] designed a pilot experiment in which they compared the effect of three doses
of curcumin (200, 400, and 1000 mg) on inflammatory serum cytokines in order to avoid the use of
animal models. These authors concluded that the optimal dose would be 400 mg/day. Furthermore,
Nicol et al. [28] selected a dose of 5000 mg/day of natural curcumin based on a study in mice, which
resulted in a dose of less than 5% bioavailable curcumin (<200 mg/day of bioactive curcumin). Finally,
Basham et al. [32] selected a dose of 1500 mg (69 mg of curcuminoids). This study implemented a newly
enhanced absorption and pharmacokinetics of fresh turmeric derived from curcuminoids in comparison
with the standard curcumin from dried rhizomers. Dosing was determined via the manufacturer’s
recommendations [34]. However, most studies investigating curcumin supplementation have utilized
dosages ranging from as little as 50 mg/day [35] to 2.5 g/day [28], demonstrating efficacy and safety in
humans. Further, internal quality control testing was performed by the manufacturer, ensuring safety
and authenticity of the supplement.
Thus, while research using curcumin supplements with improved bioavailability (Meriva
Curcumin [4,5]; Theracurcumin Theravalues [1,24,25,36]; Longvida [30]; CurcuWIN [10];
CurcuFresh [32] determined the dose at values between 150 and 1500 mg/day, studies using curcumin
naturally [28,31] needed higher doses (5000 to 6000 mg/day) to achieve similar bioavailability.
The Korean Food and Drug Safety Administration has declared turmeric safe and tolerable in
humans, and long-term studies with curcumin have revealed no toxic or adverse effects. However,
in a supradosing range, with higher doses than the studies described in this manuscript of between
8 and 12 g, some subjects experienced mild nausea or diarrhea [37]. One consideration that should
be taken into account in supplementation with curcumin in athletes, who are themselves susceptible
to iron deficiencies with or without anemia, is the interaction between high doses of curcumin and
the alteration of iron metabolism by the chelation of iron and elevation of hepcidin, which could be
another potential cause of decreased iron levels [38]. For this reason, piperine increases in importance
because it allows a high bioavailability of curcumin with lower doses [39]. In this sense, Delecroix et
al. [31] used curcumin (6 g) plus piperine (20 mg) per day. However, it is not possible to determine the
degree of absorption of the different formulations of curcumin because the studies do not reveal the
plasma concentrations.
We believe that curcumin can be safely used as a modulating therapy for markers of inflammation
and exercise-induced muscle damage. However, in spite of the safety of the curcumin dose, it is
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necessary to develop more precise criteria according to the type of curcumin administered, the duration
of treatment, and the type of sport performed, in order to establish an optimal dose and an effective
intake time that are capable of attenuating the effects of exercise on inflammatory responses and
muscle damage.
4.2. Exercise-Induced Muscle Damage (EIMD)

EIMD could affect different muscle dimensions such as muscle pain, muscle performance, and
muscle enzyme activity.
4.2.1. Effect on Muscle Pain

Muscle pain can be induced by EIMD or an unaccustomed activity [40] and results in discomfort
at the site of the injury and loss, among others, of muscle function and strength; hence, it limits physical
function for several days after exercise [41]. The potential effect of curcumin supplementation in
reducing muscle pain could be due to the effect it has on suppressing the induction of expression of the
isoform COX-2 [25], thus avoiding the production of mediating substances, such as prostaglandin E2
(PGE2), histamine, bradykinin, and serotonin derived from COX-2 that activate nerve endings [19].
The action of curcumin decreasing these mediators, especially PGE2, would provide the attenuation of
the phenomenon of long-lasting hyperalgesia that occurs in afferent sensory fibers of type C [42].
In this sense, ten studies [1,2,5,10,24,25,28,30–32] evaluate the ability of curcumin to attenuate
muscle pain; eight of which do so through the Visual Analog Scale (VAS) [1,2,4,10,24,25,28,32].
Concretely, Nicol et al. [28], described that the intake of 2.5 g of curcumin supplementation in
capsules taken 48 h before and 72 h after eccentric exercise caused significant reductions in pain.
Aligned with this, in two studies by Tanabe et al. [1,25], a significant reduction in muscle pain was
demonstrated by the effect of administering 180 mg of curcumin supplement (90 mg twice daily) in
Theracumin-Theravalues capsules, only when administered four [25] and seven [1] days after eccentric
contraction exercise. In addition, the curcumin supplementation (1.5 g) resulted in significantly
decreased muscle soreness overall (VAS scale 2.88) when compared to the placebo (VAS scale 3.36) (p <
0.0120) [32]. The supplementation continued for three days during the follow-up testing sessions; thus,
28 total days of supplementation were implemented. Finally, Nakhostin-Roohi et al. [24] showed that
one dose of 150 mg of curcumin supplementation in capsules (Theravalues) taken immediately after
exercise significantly reduced muscle pain at 48 and 72 h after eccentric exercise.
However, Jäger et al. [10] showed non-significant improvements in exercise-induced total thigh
soreness and indicated that the 200 mg curcumin groups reported 26%, 20%, and 8% less soreness
immediately, 24 h, and 48 h after exercise, respectively, as compared the soreness levels that were
reported in the PLA and 50 mg curcumin groups; these differences failed to reach statistical significance.
The supplementation of curcumin was for eight weeks prior to downhill running protocol VAS. In this
sense, Tanabe et al. [2] did not find a significant effect in delayed onset muscle soreness (DOMS) using
150 mg of curcumin or placebo orally before and 12 h after each eccentric exercise.
There are many possible supplementation conditions in terms of dose, frequency, and time points.
Nosaka et al. [43] reported that essential amino acid supplementation given both 30 min before and
immediately after eccentric exercise did not affect any indicators of muscle damage, but when the
supplementation was continued for next four days after exercise, it attenuated increase in muscle
soreness and range of motion (ROM). Thus, it is possible that curcumin supplementation has an effect
on DOMS if the curcumin is given on recovery days, for a period of at least 3 days, as in [28,32], or for
a longer period of time [1,25]. However, a single dose of curcumin (150 mg) immediately after exercise
significantly reduced muscle pain [24]. This situation may justify the need for a series of studies to
investigate whether curcumin supplementation for recovery immediately after exercise will provide
greater effects on muscle damage.
On the other hand, three studies [5,30,31] evaluate the ability of curcumin to attenuate muscle
pain using other scales. In particular, Drobnic et al. [5] observed a tendency towards less pain, but
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no significant improvements, in the lower extremities after 200 mg curcumin supplementation taken
48 h before and 24 h after a downhill race. In addition, Mc Farling et al. [30] investigated subjective
quadriceps muscle soreness, finding no significant difference in muscle soreness or activities of daily
living soreness between supplementation with curcumin (400 mg; 24 h before and for 72 h after) and
placebo. Moreover, Delecroix et al. [31] showed no beneficial effect of curcumin (2 g; 48 h before and
for 48 h after) in reducing muscle pain, as evaluated by the Hooper scale and subjective quadriceps
muscle soreness.
It is possible that differences in pain perception outcomes in studies that could not establish the
same effect are probably due to the subjective perception of the patient’s pain intensity. For this reason,
a combination of physiological and psychological factors could play an important role in individual
perception, thus potentially improving recovery from training [44]. Therefore, it cannot be ruled out
that perception, psychological factors, and placebo effects may have influenced the reported results.
We described that the doses of curcumin that showed benefits for muscle pain attenuation were in
a wide range (150 mg–2.5 g); however, what might be effective is administration immediately after
exercise and/or within at least 72 h after exercise.
4.2.2. Effect on Muscle Performance

Muscle damage caused by mechanical stress during eccentric exercise and subsequent
inflammatory responses lead to a deterioration of muscle performance. Thus, changes in maximum
voluntary contraction (MVC) force, range of motion (ROM), and isokinetic dynamometry reflect the
dimension and time progression of EIMD, and therefore, these parameters can be used as markers
of athletic performance [1,2,31]. MVC, isokinetic dynamometry, and ROM are diminished by the
activation of NF-κB under the high mechanical stress caused by the overuse of some joints in sport,
which generates fragments of the extracellular matrix of the bone or cartilage. These fragments are
recognized by receptors of innate immunity, which recognize pattern recognition receptors, called
toll-like receptors. Cell activation mediated by this process ends between the activation of NF-κB,
which is a stimulator of the secretion of inflammatory cytokines (IL-1, IL-1b, IL-2, IL-15, IL-21, TNF-α),
chemokines (CCL-19, CCR-7), and metalloproteases (MMP-13, ADAMSTS-4), all responsible for
producing tissue damage [45]. It is probably the action of curcumin that is responsible for minimizing
the incapacitating tissue effects, as it is a therapeutic agent that blocks the signaling pathway of
NF-κB. In addition, reducing leucocyte adhesion and migration, and as a result, relieving pain and
swelling, improves joint mobility and stiffness [16,37]. Three studies conducted by Tanabe et al. [1,2,25]
evaluated MVC and ROM. One of them [2] showed that 150 mg of curcumin both before and 12 h after
50 eccentric contractions of the elbow flexors presented a significantly smaller decreasing magnitude
on the MVC. However, ROM decreased significantly at all measurement times (24, 48, 72, and 96 h)
in the curcumin group, but there were no significant interaction effects for changes between placebo
and curcumin. Another study by the same author [1] demonstrated significantly faster recovery of
torque MVC and significant improvements in ROM only when 180 mg of curcumin was ingested after
exercise [1]. Moreover, Tanabe et al. [25] found that when 180 mg curcumin was ingested during the
four days following exercise, ROM improved significantly after three to four post-exercise days, with
no relevant changes in MVC for any of the study conditions. Recently, Jäger et al. [10] reported that
only 200 mg (CurcuWIN® ) was effective in preventing the observed decreases in peak extension torque
values seen 1 and 24 h after exercise that damaged muscles.
In this way, these studies suggest that the intake of a dose of curcumin (90–200 mg) after exercise
may be effective as a muscle performance enhancer by benefiting the recovery process.
4.2.3. Effect on Muscle Enzyme Activity

Intense exercise increases the circulating levels of markers for muscle damage such as lactate
dehydrogenase (LDH), CK, myoglobin (Mb), and transaminases (alanine aminotransferase (ALT) and
aspartate aminotransferase (AST)). All these parameters are indicative of increased EIMD and OS,
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which negatively affect athletes because they reduce exercise performance and can also put their health
at risk [46].
The effect of curcumin on CK enzyme activity levels after exercise was studied in eight
investigations included in this systematic review [1,2,5,24,25,28,30,31]. Five of them [1,2,24,28,30]
presented significantly lower maximum CK activity in the curcumin-supplemented group compared
to the placebo groups. Moreover, although there were no significant differences, three studies [5,25,31]
observed that CK levels tended to increase less in the curcumin group. Potentially, the decrease in CK
after curcumin supplementation could be attributed to an antioxidant role by neutralizing oxygen free
radicals (ROS) produced during the electron transport chain of oxidative phosphorylation, necessary
for energy requirements in physical exercise [8]. Another possible mechanism of CK’s activity reduction
may be the inhibition of the production of histamine and prostaglandin by suppressing the positive
regulation of COX-2, a pathway involved in vascular permeability [47]. In local areas with inflammation,
they reduce the permeability of the membranes, thus reducing the intracellular–intravascular flow of
CK. Thus, the limitation of vascular permeability could be the key factor in reducing inflammation
and muscle pain [48]. The differences between studies may be due to dose, timing of curcumin
supplementation, and intensity of physical activity. In addition, Nakhostin-Roohi et al. [24] showed
ALT and AST at significantly reduced levels in curcumin group.
The findings of this study [24] suggest that a 150 mg dose of curcumin, when administered
immediately after exercise, may have protective effects on muscle damage by significantly reducing the
levels of three markers of circulating muscle damage (CK, AST, and ALT). It is difficult to concretize
supplementation with curcumin because there are many possible supplementation conditions in
terms of dose, frequency, and time points where curcumin has been shown to be effective in relation
to its potential to decrease muscle damage, which is reflected in decreased CK activity in the
supplemented groups.
4.3. Effect on Inflammatory Markers

Curcumin is currently recognized for its potential anti-inflammatory effect [13,15]. In this sense,
inflammation is a physiological process in response to physical exercise. Pro-inflammatory cytokines
and chemokines produced by immune cells during the immune response interact with their receptors
by activating signaling pathways of the inflammatory response [20]. The possible positive effect of
curcumin supplementation on inflammatory response may be due to the modulating action of curcumin
on inflammatory signaling cascades. These signaling pathways include the nuclear factor κB pathway
(NF-κB), the signal transducer and transcription activator Janus kinase (JAK/STAT), and mitogen
activated protein kinase (MAPKs). Curcumin inhibits the activation of NF-κB, suppresses the activation
and phosphorylation of JAK/STAT proteins, and inhibits MAPK signaling through its interaction with
three major members of this pathway, including JNK, p38, and ERK. Bisdemethoxycurcumin suppresses
infiltration, activation, and maturation of leukocytes, and also the production of proinflammatory
mediators TNF-α, IL-8, and IL-6 at the site of inflammation. Another effect the potential of curcumin is
to act as an immunomodulator by intervening in the suppression of immune responses acquired in T
cells, inhibiting the activation, differentiation, and production of cytokines [20].
In the studies analyzed in this systematic review, the pro-inflammatory cytokines
TNF-α [1,2,28,30,32], interleukin 6 (IL-6) [2,4,28,30], and IL-8 [1,5,30] were evaluated as markers
of inflammation. In relation to TNF-α, Nicol et al. [28] and Tanabe et al. [1,2] did not observe any
effect of curcumin supplementation on this inflammation marker. However, McFarlin et al. [30] and
Basham et al. [32] reported sustained suppression of TNF-α for up to one day after exercise in the
curcumin supplemented group compared to the placebo group. Moreover, TNF-α was significantly
lower with curcumin at two days and four days and trended towards being lower with curcumin
at three days compared to placebo [30]. It is likely that the effectiveness of curcumin on the plasma
decrease of TNF-α is dependent on a minimum dose of 400 mg administered before exercise and for
72 h after exercise (at minimum), which may explain the differences with respect to the studies by
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Tanabe et al. [1,2]. In addition, commercial formulas of curcumin [30,32] implemented an enhanced
absorption and pharmacokinetics as compared to standard preparations of curcumin [28].
On the other hand, curcumin supplementation showed a downward but non-significant trend
on IL-6 cytokines derived from exercise practice [2,4,30]. Moreover, Drobnic et al. [5] and Mc Farlin
et al. [30] showed that 400 mg of curcumin supplementation (48 h before and 24–72 h after exercise)
significantly reduced IL-8 levels. However, Tanabe et al. [1] observed non-significant decreases in
IL-8 12 h after exercise, when subjects were supplemented with 180 mg of curcumin for seven days
before the eccentric exercise test. The differences in dosage and timing could be the cause of differences
in IL-8 regulation. According to these results, it is possible that a minimum of 400 mg of curcumin
administered before and for at least 24 h after exercise may be necessary.
In this way, the promoters of IL-6 and IL-8 cytokines possess binding sites for NF-κB, C/EBPβ,
and c-Jun [49]. We believe the role of NF-κB inhibition is a therapeutic objective of curcumin in
inflammation because of the importance of NF-κB for the regulation of the constitution and expression
of IL-6 and IL-8 [49,50]. McFarling et al. [30] concretely observed that significant inhibition of NF-κB
could be related to a significant decrease in IL-8 and a downward trend in IL-6. Thus, supplementation
with 400 mg of curcumin, two days before and three days after exercise, appears to be effective in
attenuating exercise-induced inflammation because of its direct action on NF-κB, which influences the
cytokines IL-8 and IL-6.
Other cytokines, such as IL-10 [4,30], with anti-inflammatory properties capable of inhibiting the
synthesis of pro-inflammatory cytokines and IL1RA [4], which is a key mediator in the inflammatory
response, were evaluated. Lower levels of non-significant IL-10 and IL1RA were reported in the
curcumin-administered groups as compared to the placebo group.
4.4. Effect on Oxidative Markers

This mechanical stress derived from exercise triggers an inflammatory response and the production
of ROS. ROS are able to maintain inflammation and in general a high degree of oxidative stress by
stimulating the activation pathways of transcription factors such as nuclear factor-κB (NF-κB), a
pro-inflammatory master switch that controls the production of inflammatory energy, and are involved
in cell damage [32]. This sustained inflammatory response and the high degree of oxidative stress
lead to the accumulation of neutrophils and increased “inflammatory growth medium” production of
oxidative enzymes, cytokines, and chemokines [15]. In every organism, there is a protection system
formed by antioxidant compounds and enzymes that participate in the transformations of these
species. One of them is the enzyme glutathione peroxidase (GPx). This enzyme uses glutathione to
reduce peroxides, thereby protecting membranes and other cellular structures from the action of lipid
peroxides and free radicals [51]. In addition, catalase (CAT) is one of the enzymes involved in the
destruction of hydrogen peroxide generated during cell metabolism [52]. The overproduction of ROS
eventually surpasses the body’s antioxidant capacity [15].
Against this background, curcumin could be useful because it has been described as suppressing
the activation of NF-κB and the promotion of antioxidant response by the transcription activation of
Nrf2, which could neutralize these harmful effects related to ROS [18]. Drobnic et al. [5] observed
non-significant increases in GPx and CAT after supplementation with curcumin (200 mg) before and
after exercise. Other antioxidant markers, such as reactive metabolites of oxygen serum (d-ROMs)
and potential biological antioxidant (BAP) concentrations, were not different between curcumin and
placebo trials after supplementation with curcumin (90 mg) before and after exercise [1]. In agreement
with this, Basham et al. [32] found that exercise was not different between curcumin and placebo trials
in total antioxidant capacity (TAC), and that malondialdehyde is formed by the lipid peroxidation of
unsaturated fatty acids and is a marker of oxidative degradation of the cell membrane. It is difficult to
determine the efficacy of the antioxidant capacity of curcumin, considering the above results. However,
in this study, TAC remained significantly higher in the curcumin group after exercise compared with
the levels in the placebo group (p < 0.05) [24]. The findings of this study suggest that a 150 mg dose
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of curcumin may have antioxidant effects. The differences between the studies could be due to the
timing of ingestion of curcumin, manifesting only a significant effect when ingested immediately after
exercise, as suggested by Nakhostin-Roohi et al. [24].
5. Limitations, Strengths, and Future Lines of Research

The main limitations of the present systematic review are related to the low number of studies
investigated on this subject and to the fact that most of them had a relatively small number of
participants. Because of this, it is essential to take into consideration that the studies analyzed
were conducted in populations with different levels of physical activity and using different research
protocols, which increases the heterogeneity between studies. In addition, dosage, duration of
treatment, and formulation of curcumin were not uniform across investigations, which could affect
some of the results, particularly because of the limitation associated with the bioavailability of the
supplement. Likewise, in some of the selected studies, specific diet controls were carried out prior to
the study, telling the participants not to eat foods containing curcumin (e.g., curry) or to follow any
nutritional guidelines to avoid the main polyphenols in the diet. However, in other studies, only the
consumption of anti-inflammatory drugs was limited, whilst it is still possible that some habitual foods
produce anti-inflammatory effects. Finally, it should be noted that the studies analyzed only evaluated
inflammatory markers from blood samples, but none in muscle tissue.
The development of curcuminoids in nanoformulations to improve the pharmacokinetics,
bioavailability, and biological activity of curcuminoids is currently under investigation [53]. Further
research is needed to determine whether these new formulations might be more effective in treating
inflammation and the time course of exercise-induced muscle damage. Another way to improve
bioavailability would be through piperine, as demonstrated by Delecroix et al. [31], who included 6 g
of curcumin +60 mg of piperine/day. Piperine is a thermonutrient that exerts its thermogenic action
on the epithelial cells of the small intestine, increasing the rate of nutrient absorption and therefore
increasing its bioavailability [54].
Although the studies used in this review were not conducted exclusively in a competitive sports
population, they provide an adequate justification to support broader research that could eventually
confer its acceptance as a standard in the recovery of muscle function and inflammatory parameters in
the framework of physical activity. Furthermore, a strength of this systematic review is quality control
through PRISMA and Mc Master.
6. Practical Applications
In general, curcumin supplementation could be used during periods of high demand, tournaments,
or competitive events to speed up the recovery of muscle function and counteract the size and progress
of symptoms associated with exercise-induced muscle pain [14]. In addition, curcumin has been
used in various protocols before and/or after exercise, to decrease inflammation and muscle damage
through its ability to modulate the inflammatory response and its antioxidant effect. However,
implementing curcumin as an ergogenic aid should be considered in light of the sporting objective,
since the persistent use of anti-inflammatory substances that benefit recovery could affect training
adaptations [55]. However, maximizing resilience at the expense of training adaptations may be
desirable in athletes in competitive seasons.
Since the athlete’s recovery in a period of high training or competition loads is limited to a
specific moment, the decision on the supplementation strategy to be implemented should be made
considering the physiological effects of the substance. Along these lines, it could be considered that
supplementation with curcumin in combination with one or more substances that act on different
physiological mechanisms could result in a synergistic effect on the parameters of inflammation and
muscle damage in the recovery process.
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Finally, curcumin should always be used as a pharmaceutical preparation and not as a spice to
avoid the toxic effects of fungical aphlotoxins. In addition, caution is needed with athletes who are
sensitive to gastric irritation.
7. Conclusions
In summary, the use of curcumin reduces the subjective perception of the intensity of muscle pain.
Likewise, curcumin is able to decrease muscle damage through the reduction of muscle CK activity
and to increase muscle performance. Moreover, supplementation with curcumin exerts a post-exercise
anti-inflammatory effect by modulating the pro-inflammatory cytokines TNF-α, IL-6, and IL-8, and
curcumin may have a slight antioxidant effect. The minimum optimal dose to achieve a positive
impact would be recommended doses between 150 and 1500 mg/day, when administered before and
immediately after exercise, and for 72 h after. Finally, curcumin should only be recommended to
athletes who are willing to use ergogenic aids to increase performance, and it should be recommended
only on an individual basis to modulate some of the muscle damage and inflammation caused by
physical activity. Oral curcumin supplementation has been shown to be effective pre and/or post
physical activity.
Author Contributions: D.F.-L.: conceived and designed the investigation, analyzed and interpreted the data,
drafted the paper, and approved the final version submitted for publication C.I.F.-L. and J.M.-A.: analyzed and
interpreted the data, critically reviewed the paper and approved the final version submitted for publication. J.S.C.
and A.C.M. and A.C.G.: critically reviewed the paper and approved the final version submitted for publication.
All authors have read and agreed to the published version of the manuscript.
Funding: The authors declare no funding sources.
Acknowledgments: The authors are grateful to the Foundation Institute of Health Sciences Studies of Castilla-León
(ICSCYL) for its collaboration in infrastructures, bibliographic bases and computer support.
Conflicts of Interest: The authors declare no conflict of interest.
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20
nutrients
Article
The Effects of Low- and High-Glycemic Index Sport
Nutrition Bars on Metabolism and Performance in
Recreational Soccer Players
Mojtaba Kaviani 1,2, *, Philip D. Chilibeck 2, *, Spencer Gall 2 , Jennifer Jochim 2 and
Gordon A. Zello 3
1 School of Nutrition and Dietetics, Faculty of Pure & Applied Science, Acadia University, Wolfville,
Nova Scotia, NS B4P 2R6, Canada
2 College of Kinesiology, University of Saskatchewan, 87 Campus Dr, Saskatoon, SK S7N 5B2, Canada;
[email protected] (S.G.); [email protected] (J.J.)
3 College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, SK S7N 5E5,
Canada; [email protected]
* Correspondence: [email protected] (M.K.); [email protected] (P.D.C.);
Tel.: +1-902-585-1884 (M.K.); +1-306-966-1072 (P.D.C.)
Received: 23 February 2020; Accepted: 31 March 2020; Published: 2 April 2020
Abstract: Consumption of low-glycemic index (GI) carbohydrates (CHO) may be superior to high-GI
CHO before exercise by increasing fat oxidation and decreasing carbohydrate oxidation. We compared
the effects of pre-exercise feeding of a low-GI lentil-based sports nutrition bar with a high-GI bar on
metabolism and performance during a simulated soccer match. Using a randomized, double-blind,
counterbalanced, crossover design, participants (n = 8) consumed 1.5 g/kg available CHO from a
low-GI bar (GI = 45) or high-GI bar (GI = 101) two hours before a 90 min simulated soccer match, and
0.38 g/kg body mass during a 15 min half-time break. The test involved alternating 6 min intervals of
paced jogging, running, walking, and sprinting, and 3 min intervals of soccer-specific skills (timed
ball dribbling, agility running, heading, kicking accuracy). Carbohydrate oxidation rate was lower
during the match after consuming the low-GI compared to high-GI bar (2.17 ± 0.6 vs. 2.72 ± 0.4 g/min;
p < 0.05). Participants performed better during the low-GI versus high-GI bar condition on the
agility test (5.7 ± 0.4 versus 6.1 ± 0.6 s; p < 0.01) and heading (i.e., jumping height 24.7 ± 4.3 versus
22.2 ± 4.5 cm; p < 0.01) late in the soccer match (72 min). A low-GI lentil-based sports nutrition bar
provides a metabolic benefit (lower carbohydrate oxidation rate) and a modest improvement in agility
running and jumping height (heading) late in the test.
Keywords: Carbohydrate; high-intensity exercise; fatigue
1. Introduction
Carbohydrate (CHO) is an important source of energy throughout strenuous prolonged exercise.
Premature fatigue during prolonged exercise is linked with depletion of carbohydrate stores (i.e., blood
glucose and liver and muscle glycogen stores). Thus, carbohydrate consumption before and during
exercise improves exercise performance compared with a fasted condition [1,2]. Muscle glycogen
concentrations are directly correlated to time to fatigue during moderately strenuous exercise ranging
from 60%–80% of maximal oxygen uptake (VO2max ) [1]. Thus, endurance and high-intensity intermittent
exercise will be adversely affected by reduced glycogen stores. During soccer matches, this would most
likely occur in the second half of a game [3–5]. Soccer players with lower levels of muscle glycogen
cover less distance and run at lower speeds during the last 15 min of a match [6]. Total number of
sprints and markers of acceleration and deceleration capacity are reduced in the last 15 min of the
21
Nutrients 2020, 12, 982
normal duration of a soccer match [7]; therefore, research that targets maintaining these performance
outcomes during a soccer match is important.
The glycemic index (GI) differentiates types of carbohydrates based on how fast they cause an
increase in blood glucose concentrations [8]. Some studies have indicated consumption of low-GI foods
prior to exercise may improve exercise performance compared to high-GI foods [9,10]. Low-GI foods
cause a lower insulemic response compared to high-GI foods. Insulin inhibits fat oxidation during
exercise [11]; therefore, consumption of low-GI foods might allow increased utilization of fats, lower
carbohydrate usage, and preservation of glycogen stores [12–14]. Advantages of carbohydrate ingestion
with different GIs prior to prolonged endurance exercises are well documented [15–17]; however,
further studies need to address the possible impact of foods with different GIs on high-intensity
intermittent exercise, important for many team sports (e.g., soccer, hockey, rugby). It is important to
note that, during low to moderate intensity intervals (e.g., rest and recovery times) of high-intensity
intermittent exercise, a considerable amount of energy needed for exercising muscles is provided by fat
oxidation [18,19]. Our previous studies have shown some metabolic benefits (i.e., lower insulin levels,
higher fat oxidation, lower carbohydrate oxidation and reduced lactate levels) when low-GI meals
are consumed before interval treadmill exercise programmed to simulate the repeated high-intensity
intervals of a typical soccer match, but performance specific to soccer is difficult to evaluate on a
treadmill [20–22]. In these previous studies, boiled lentils were compared to high-GI foods (i.e., mashed
potatoes with egg whites added to match for protein), but these meals may not be typical before
matches for soccer players (or other athletes involved in sports with high-intensity intervals). Endurance
athletes often consume sport nutrition bars [23] and surveys of youth soccer players indicate that about
37% consume food, such as sport nutrition bars, up to 1 h before games in an attempt to improve
performance [24]; however, the effectiveness of a sport nutrition bar for soccer performance has never
been evaluated. From a practical point of view, using sport nutrition bars (high-CHO) can be considered
when time is limited before the start of competition. Therefore, the purpose of the current study was to
evaluate low- and high-GI sport nutrition bars, consumed before and at half time on metabolism and
performance during a soccer-specific field test, which incorporates skills important for soccer performance
(i.e., agility running, ball dribbling, kicking accuracy, and ball heading) [25]. We hypothesized that a
low-GI sport nutrition bar would be superior to a high-GI sport nutrition bar to improve performance
and metabolic responses when consumed before and during a simulated soccer match.
2. Materials and Methods
2.1. Participants
Eight male recreational soccer players participated in this study. Their mean ± standard deviation
for age, body mass, and predicted maximal oxygen uptake values were 30 ± 7 years, 76.6 ± 8.6 kg,
and 56.5 ± 2.5 mL/kg/min, respectively. The University of Saskatchewan Biomedical Research Ethics
board approved the study protocol, and all participants signed a consent form before the study began.
The approval number is 12–33. The approval date was February 21, 2012.
2.2. Study Design

Using a randomized, double blind, counter-balanced cross-over design, low-GI and high-GI
sport nutrition bars were consumed two hours before and at half time of a simulated soccer match
during which soccer-specific skills (agility, ball dribbling, heading, and kicking accuracy) were assessed.
Plasma glucose and insulin, non-esterified free fatty acids (NEFA), as well as fat and carbohydrate
oxidation were assessed before and during the simulated soccer match.
2.3. Preliminary Test

Participants initially had their maximal aerobic power (VO2max ) estimated by a shuttle-run
test that involved running 20 m while the speed increased by 0.14 m/s each minute until volitional
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Nutrients 2020, 12, 982
exhaustion [26]. The purpose of this test was to determine participants’ aerobic fitness and to determine
maximal running velocity, which was used to set speeds during the exercise tests used to evaluate the
different feeding conditions. Participants then performed a familiarization test of the simulated soccer
match. This test was identical to the test they performed during the sports bar conditions, but was
used as a “practice” run. The purpose of this practice run was to minimize any learning effects from
one test to another. The practice trial involved performing 10 six-minute sessions of running between
two cones that were 20 m apart, where speed was alternated between sprinting, running, jogging,
and walking to simulate the exercise performed during an actual soccer match [21,25]. Speeds of
walking, jogging, and running were adjusted according to each participant’s predicted maximal aerobic
power [26]. The speeds were dictated by “beeps” emitted from a sound system that indicated when the
participant was required to reach the next 20 m distance. The speed of walking, jogging, and running
were set at 25%, 55%, and 95%, respectively, of the maximal speed reached in the initial maximal
shuttle run test [27]. The Bitworks Team Beep Test software (Version 4.1, Bath, UK) was used to write
the scripts for each participant. Each 6 min block alternated 60 m of jogging, running, and walking,
and 20 m of sprinting. Performance was assessed by tests of either agility running/ball dribbling or
kicking/heading of a soccer ball. These pairs of performance tests were alternately performed between
the 6 min jogging-running-walking-sprinting sessions with the exception of the first and last 3 min
periods of the test during which all four performance tests were completed [25]. The total time of the
exercise test (i.e., the 10 six-minute intervals and the testing between intervals) was approximately
90 min which is the same duration as an actual soccer match. The study diagram is shown in Figure 1.
This soccer test has been shown to be highly reproducible and is sensitive to improvement with
carbohydrate feeding [25]. Separate performance scores were derived for agility, dribbling, kicking
accuracy, and heading (Figure 2). Time to complete the agility and ball dribbling courses were recorded
for assessment of performance of these tests. The highest vertical jump was recorded for heading
performance. A vertical jump measuring device (Vertec, Power Systems (PS), LLC, Knoxville, TN,
USA) was used; participants were instructed to use their heads rather than their hands to reach the
vanes. Kicking accuracy was scored according to targets set up on a wall net.
23
Nutrients 2020, 12, 982
24
Figure 1. Schematic of the simulated soccer match. “#REF” denotes fingertip blood collection time points for glucose.
Nutrients 2020, 12, 982
1.83m
1 3 1
9.14m 10m 3.5m

0.91m
3 5 3
1 3 1
2.74m
2.74m
Figure 2. Schematic of the scoring grids for kicking accuracy (left), ball-dribbling (middle), and agility
(right) protocols. Arrows represent the distance between the cones. Adopted from Currell et al., [25].
2.4. Experimental Test

Participants reported to the lab on two different occasions after a 12 h fast for a low-GI lentil-based
bar (Genki Foods Inc., Winnepegosis, MB, Canada) test and a high-GI bar (Clif Bar Inc. Berelely, CA,
USA) test. Each condition was separated by at least one week. The GI of the lentil bar was 45 [28] and
the GI of the Clif bar was 101 [29]. The crunchy peanut butter flavor Clif bar was used because it most
closely matched the Genki Bar for macronutrients and calories. The characteristics of the nutrition
bars for a 70 kg participant are shown in Table 1. The testing was double blind, that is, neither the
participant nor the researchers knew what type of nutrition bar was consumed. The blinding was
achieved by having a separate research assistant prepare the food and having the participant consume
the food in an isolated room two hours before the exercise session. Wrappers were removed from the
bars and an appropriate amount of bar was placed in plastic bags. The high- and low-glycemic index
sport nutrition bars were similar in appearance (i.e., same color and consistency).
Table 1. Characteristics of the sport nutrition bars for a 70 kg participant.
Description Low-GI High-GI

Energy (kcal) 758 761
Fat (g) 19 20
Total Carbohydrate (g) 127 116
Available carbohydrate (g), i.e.,
105 105
Total carbohydrate minus fiber
Protein (g) 39 31
Glycemic index 45 101
GI, glycemic index.
On each testing day participants were given enough sports bars to consume 1.5 g/kg body mass
available CHO, an amount of carbohydrate that was expected to improve performance when given
two hours before high-intensity intermittent exercise [20]. This amount is also within the range of
recommended CHO intake prior to endurance exercise performance [8]. Participants also consumed
0.38 g/kg available CHO from the bars at half time of the simulated soccer match. Participants had
20 min to consume the bars before the match and 15 min during half time. Furthermore, the exact
amount of water consumption was documented in the first trial and then it was replicated in the second
trial to minimize the impact of hydration status. The feeding and the simulated soccer match were
separated by two hours. Blood glucose was assessed by fingertip sampling before the food consumption
and at 5, 15, 30, 60, 90, and 120 min after consumption. Blood samples from an antecubital vein were
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Nutrients 2020, 12, 982
taken immediately before, at half time, and after finishing the simulated soccer match for assessment
of insulin and non-esterified free fatty acid (NEFA) levels. Fingertip blood samples were collected to
assess glucose by using a glucose meter (AccuCheck Compact Plus Sarstedt, Nümbrect, Germany).
Venous blood samples were maintained in 10 mL tubes (BD Vacutainer SST) for 30 min to clot. The
serum was then separated by centrifugation for 15 min at 3500 rpm and stored at −80 ◦ C. Insulin
concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) according
to the manufacturer’s directions (STELLUX®Chemi Human Insulin, Alpco Diagnostics, Salem, MA,
USA). The serum NEFA assay was performed using a protocol with an oleic acid standard solution as
per the manufacturer’s directions (NEFAHR (2), Wako Diagnostics Inc., Richmond, VA, USA). The
intra-assay coefficient of variations (CVs) for the insulin, and NEFA assays were <10%. Fingertip blood
samples were taken after every second 6 min exercise interval during the simulated soccer match to
measure glucose and lactate levels. Blood lactate measurement was assessed using BM-Lactate test strips
and the Accutrend®Lactate analyzer (Roche Group; Mannheim, Germany). The K4 b2®(Cosmed USA,
Chicago, IL, USA), a portable gas exchange system was used to measure oxygen consumption (VO2 ),
and carbon dioxide output (VCO2 ). Respiratory gases were collected during every second 6 min exercise
interval to estimate carbohydrate and fat oxidation. Carbohydrate and fat oxidation rates were estimated
from VO2 and VCO2 by using stoichiometric equations [30]. Rating of perceived exertion, using the
modified 10-point Borg scale, was collected after each 6 min interval [31].
2.5. Dietary and Physical Activity Monitoring

Participants recorded their dietary intake and physical activity for the 24 h before the feeding
conditions. These were photocopied and given back to the participants so they could duplicate their
diets and physical activity levels during subsequent feeding conditions. This ensured that participants
arrived for each exercise feeding condition with similar diets and exercise the previous 24 h.
2.6. Statistical Analysis

All variables were analyzed with a two-factor repeated measures analysis of variance (ANOVA)
with factors for food condition (low-GI lentil bar vs. high-GI Clif Bar) and time during the exercise test.
When there was a time main effect or an interaction between condition and time, a Least Significant
Difference (LSD) post-hoc test was used to determine differences between pairs of means. All variables
were also assessed for order effects with a two-factor ANOVA with factors for order (first condition vs.
second condition) and time during the exercise test. Significance was accepted at a p-value less than
0.05. All results are reported as means and standard deviations.
3. Results
3.1. Blinding, Order Effects, and Adverse Events

When queried as to which bar condition participants thought they had consumed, two participants
guessed correctly, while all others responded that they were unsure; this indicated the success of the
blinding. There were no order effects or order*time interactions for any of the outcome variables
(p > 0.05). There were no adverse events associated with the study. None of the participants complained
of gastrointestinal discomfort after consumption of the bars.
3.2. Glucose and Insulin Responses

There was a condition*time interaction for glucose and insulin. The high-GI condition resulted in
higher glucose concentrations than the low-GI condition at 105, 90, and 60 min before the simulated
soccer match (p < 0.05; Figure 3A). The insulin response was higher in the high-GI condition compared
to the low-GI condition at two hours after bar consumption (p < 0.05; Figure 3B).
26
Nutrients 2020, 12, 982
3.3. Serum NEFA and Substrate Oxidation

NEFA concentration was not different prior to the exercise test in low-GI vs. high-GI conditions
(Figure 4). NEFA concentrations significantly increased in the low-GI and high-GI conditions at 45 min
and 90 min of exercise (time main effect, p < 0.05; Figure 4). No significant difference was seen between
the conditions. During the low-GI condition, carbohydrate oxidation was significantly lower compared
to the high-GI condition (p < 0.05; Figure 5A). There was no difference between conditions for fat
oxidation (p = 0.14; Figure 5B). There was a time main effect (p < 0.05) for fat oxidation with higher
rates at 45–51 min versus 63–69 min and 81–87 min (p < 0.05). No significant difference was observed
for lactate concentrations between the two conditions (Figure 6). There was a time main effect (p < 0.01)
for lactate, with values increasing at all time-points, except at 54 min (i.e., after half time) compared to
baseline (p < 0.05). The lactate at 54 min was lower than at 45 min and 90 min (p < 0.05).
3.4. Skill Performance and Rating of Perceived Exertion

There were condition*time interactions for the agility and heading tests (p < 0.05). A significant
improvement on the agility test and vertical jump height during simulated heading late in the soccer
match (72 min) was observed after consuming the low-GI versus high-GI bar (Table 2; p < 0.01).
No differences were apparent between bar conditions for skills performance of ball dribbling or kicking
accuracy (Table 2). There was a time main effect for rating of perceived exertion (RPE; increasing
throughout the simulated soccer match; p < 0.05); however, no significant difference was observed
between the two conditions (mean RPE throughout the 90 min soccer match: low-GI = 5.2 ± 1.5 versus
high-GI = 5.4 ± 1.3; p > 0.05).
30
27 Low-GI
24 High-GI
21
Insulin (μIU/mL)
18
15
12
9
6
3
0 Ύ
Pre 45 90
Time (min)
(B)
Figure 3. (A) Plasma glucose concentrations before and during the simulated soccer match, (B) Insulin
concentration before, at half time, and at the end of the simulated soccer match (* p < 0.05 low-GI vs.
high-GI sport nutrition bar). “Pre” denotes fingertip blood sample collection prior to consumption of
the sport nutrition bars.
27
Nutrients 2020, 12, 982
Figure 4. Non-esterified fatty acid concentrations before, at half time, and at the end of the simulated
soccer match for low- and high-GI conditions. Values are means ± standard deviation (SD). Time main
effect (p < 0.05) with 90 min > 45 min > pre (p < 0.05). “Pre” denotes venous blood draw prior to the
simulated soccer match.
Figure 5. (A) Rate of carbohydrate oxidation, (B) and fat oxidation during the simulated soccer match in
the low glycemic index and high glycemic index conditions. Values are means and SD. * Main effect for
condition for carbohydrate oxidation, with the low-GI condition lower than the high-GI condition; p < 0.05.
There was a time main effect (p < 0.05) for fat oxidation (45–51 min > 63–69 min, 81–87 min; p < 0.05).
Figure 6. Lactate concentrations before and during the simulated soccer match between low- and
high-glycemic index conditions. Values are means ± SD. Time main effect (p < 0.05) with all values
except 54 min >pre, and 45 min, 90 min >54 min (p < 0.05).
28
Table 2. Performance variables during the simulated soccer match.
Condition Low-GI High-GI

Time (Min) 12 30 45 54 72 90 12 30 45 54 72 90
Ball Dribbling (s) 14.0 ± 2.6 12.8 ± 2.3 11.8 ± 2.0 12.2 ± 2.2 12.6 ± 1.9 12.3 ± 2.1 13.8 ± 2.6 13.1 ± 2.3 12.1 ± 2.2 12.6 ± 1.9 12.1 ± 2.4 12.7 ± 2.7
Heading (cm) 20.5 ± 6.5 24.4 ± 4.1 24.1 ± 4.4 23.3 ± 3.5 24.7 ± 4.3 * 23.3 ± 0.9 20.3 ± 7.9 23.8 ± 4.8 23.5 ± 5.2 22.5 ± 5.2 22.2 ± 4.9 22.5 ± 5.1
Kicking (arbitrary units) 10.0 ± 5.7 11.8 ± 3.0 11.1 ± 5.5 13.1 ± 7.1 12.4 ± 4.0 9.1 ± 4.3 10.8 ± 6.5 10.1 ± 5.4 9.3 ± 5.9 9.0 ± 2.3 12.1 ± 4.5 12.8 ± 6.2
Nutrients 2020, 12, 982
Agility (s) 6.1 ± 0.4 6.0 ± 0.6 6.0 ± 0.6 5.9 ± 0.5 5.7 ± 0.4 * 6.0 ± 0.6 6.1 ± 0.4 6.1 ± 0.6 5.9 ± 0.6 6.1 ± 0.5 6.1 ± 0.6 5.8 ± 0.6
Values are means and standard deviation (SD). * Significantly different in the low-GI versus high-GI condition (p < 0.01).
29
Nutrients 2020, 12, 982
4. Discussion
The main finding of this study was that a low-GI sport nutrition bar consumed two hours before
and at half time during a simulated soccer match elicited lower carbohydrate oxidation throughout the
match and improvements in agility performance and heading (i.e., vertical jump height) late in the
match compared to a high-GI sport nutrition bar. In line with this potential for CHO supplementation
to improve performance, in a systematic review, Russell and Kingsley stated that six out of eight
included studies found that CHO ingestion in the form of 6%–8% solution of glucose, sucrose, or
maltodextrin (which would have a high-GI; i.e., GI > 70 [8]) was linked with an improvement of at
least one aspect of soccer skill performance [32]. However, to the best of our knowledge this is the
first study to address the influence of low- and high-GI sport nutrition bars consumed shortly prior to
prolonged, high-intensity, intermittent exercise, which is typical for many team sports. Although we
found improvements in some performance measures with the low-GI sport nutrition bar condition
at the 72 min time point, this did not persist to the 90 min time point of the simulated soccer match
(Table 2). This may be due to lack of adequate statistical power, or perhaps the GI of the bar consumed
does not make a difference this late in the match (i.e., glycogen depletion may be at a low enough level
in both conditions to impair performance).
Our metabolic findings are in agreement with our previous work with soccer players. In our
previous work, we showed that low-GI foods (i.e., lentils; with GI ranging from 29–36; where low-GI is
defined as < 55 [8]) consumed before a simulated soccer match on a treadmill reduced carbohydrate
oxidation [20], increased fat oxidation [21], and tended to reduce glycogen usage [22] compared to
conditions where high-GI foods (i.e., instant mashed potatoes, white bread, and egg whites to match for
protein; GI ranging from 75–81; where high-GI is defined as > 70 [8]) were consumed before exercise.
In these previous studies we did not see any difference between low-GI and high-GI conditions when
performance was evaluated by repeated sprints at the end of the simulated treadmill test. The current
study used skill performance that was quite different from these previous studies and more specific
to soccer performance. The current study also used a dietary condition (i.e., sports nutrition bars)
which is more likely to be used by soccer players before matches when less time is available for food
consumption [5].
In line with our previous studies [20,22] glucose concentrations were significantly higher in the
high-GI condition in the first 60 min following consumption versus the low-GI condition. Consequently,
insulin response in the high-GI condition was higher than the low-GI condition, which might explain
the higher carbohydrate oxidation during the test in the high-GI condition. Insulin inhibits fat
oxidation, necessitating greater carbohydrate oxidation and potentially greater glycogen usage [11].
Muscle glycogen is a major substrate during prolonged intermittent high-intensity exercise to provide
high rate of ATP re-synthesis [3,4,33]. In this study, the carbohydrate oxidation rate was lower in
the low-GI condition compared to the high-GI condition averaged across all time points (i.e., there
was a “condition” main effect), and the potential for glycogen sparing might have contributed to
improved exercise performance (i.e., agility running and heading) late in the simulated soccer match.
In line with this, Saltin [6] showed that total walking distance and sprinting speed were reduced in
soccer players with lower glycogen content versus those with higher glycogen content late in a soccer
match. Bendiksen et al. [34] reported that utilization of muscle glycogen was significantly lower in the
last 30 min of a match suggesting an important role of sustained availability of glucose later in the
match. It should be noted, however, that we did not directly assess muscle glycogen in the current
study; therefore, we cannot make conclusions on whether there was sparing of glycogen during the
low-GI condition.
It has been postulated that a reduction in lipolysis rate, and therefore NEFA, will occur following
high-GI pre-exercise meals [15,35]; however, we found no significant difference between low-GI and
high-GI conditions for appearance of NEFA in the blood. This discrepancy might have been related to
the greater intensity of our test protocol versus the other studies. During higher intensity exercise,
plasma NEFA concentrations decrease while glucose and glycogen utilization increase in skeletal
30
Nutrients 2020, 12, 982
muscle [36]. A limited rate of fat oxidation is thought to be connected with a lower flux of long
chain NEFA across the mitochondrial membrane [37,38]. An alternative explanation to the lack of
difference in NEFA concentration between conditions may be that the high-GI condition resulted in
lower intramyocellular lipids utilization rather than lower lipolysis from adipose tissue.
The main limitation of our study was the assumption that reduced carbohydrate oxidation would
lead to sparing of muscle glycogen. Direct analysis for glycogen levels by muscle biopsy would
strengthen future studies comparing high- versus low-GI foods. We attempted to standardize glycogen
levels between trials by having participants match their dietary intake and physical activity levels
the day before each trial. For better control, it would be preferable to provide standardized meals to
participants the day before trials. We would expect an increase in NEFA release from adipose tissue
with the lower insulin concentration in the low-GI condition, but this was not observed. A limitation is
that we did not assess glycerol, which may give more precise data on lipolysis. Another limitation is
that we tested athletes after an overnight fast. The typical practice of a soccer player would most likely
be to have a small breakfast and then consume a small amount of carbohydrate before the soccer match.
We supplied enough of the bars to provide 1.5 g/kg available carbohydrate before the soccer match,
an amount of carbohydrate that is recommended for improvement in endurance performance [8].
This required consumption of approximately five bars by each participant which totaled approximately
760 kcal (Table 1). We felt the addition of a breakfast before the bar consumption would result in excess
fullness in participants. Additional limitations include a relatively small participant number, and the
fact that our soccer match was simulated, rather than being an actual soccer match. Future studies
could focus on the effects of consuming the bars before actual soccer matches.
5. Conclusions and Practical Application

Previous studies carried out in our lab using treadmill protocols to measure soccer performance
(i.e., 1 min intervals of high-intensity running at the end of a simulated soccer match) were not very
specific to soccer performance. This motivated the use of a field simulated soccer test incorporating
soccer skills (i.e., agility, dribbling, kicking, and heading) to optimize the specificity of the test to the
sport of soccer. Another novel aspect of the study was the assessment of sport nutrition bars given with
adequate amount of recommended available carbohydrates (i.e., 1.5 g/kg) before endurance exercise
performance. Previous research has generally shown that consumption of sports nutrition bars has no
effect on endurance exercise performance [39–41]; however, these studies evaluated the effect of only a
single sports nutrition bar before exercise. This would deliver well below the recommended amount of
available carbohydrate for improving exercise performance; therefore, consumption of higher number
of sports nutrition bars might seem practical. Sport nutrition bars containing high-CHO can act as an
immediate snack, in particular, when soccer players are under time constraints before matches.
In conclusion, a low-GI sport nutrition bar consumed before a simulated soccer match elicited a
lower carbohydrate oxidation rate and a modest improvement in performance (i.e., better agility and
heading performance late in a simulated soccer match) versus a high-GI sport nutrition bar. Further
studies are required to investigate how sport nutrition bars varying in GIs could impact soccer skill
performance during prolonged match play (i.e., over/extra time, penalty kicks) when carbohydrate
stores will be further depleted.
Author Contributions: The study was designed by P.D.C. and G.A.Z.; data were collected by M.K., J.J., and S.G.;
data interpretation, analysis, and manuscript preparation were undertaken by M.K., P.D.C., and G.A.Z. All authors
approved the final version of the paper. All authors have read and agreed to the published version of the manuscript.
Funding: This study was funded by Agri-food Canada and the Saskatchewan Pulse growers. The sponsors had
no role in the design, execution, interpretation, or writing of the study.
31
Nutrients 2020, 12, 982
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33
nutrients
Article
Application of Continuous Glucose Monitoring for
Assessment of Individual Carbohydrate Requirement
during Ultramarathon Race
Kengo Ishihara 1,2, *, Natsuki Uchiyama 1 , Shino Kizaki 1 , Emi Mori 1,3 , Tsutomu Nonaka 4
and Hiroshi Oneda 5
1 Department of Food Sciences and Human Nutrition, Faculty of Agriculture, Ryukoku University,
Shiga 520-2194, Japan
2 Department of Life Science, Manchester Metropolitan University, Manchester M1 5GD, UK
3 Department of Food and Nutrition, Jin-ai Women’s College, Fukui 910-0124, Japan
4 Tail Ender’s Trail Running Life, Tokyo 176-0004, Japan
5 Nagatasangyo Co., Ltd, Shiso 671-2544, Japan
Received: 6 March 2020; Accepted: 14 April 2020; Published: 17 April 2020
Abstract: Background: The current study intended to evaluate the feasibility of the application of
continuous glucose monitoring to guarantee optimal intake of carbohydrate to maintain blood glucose
levels during a 160-km ultramarathon race. Methods: Seven ultramarathon runners (four male and
three female) took part in the study. The glucose profile was monitored continuously throughout
the race, which was divided into 11 segments by timing gates. Running speed in each segment was
standardized to the average of the top five finishers for each gender. Food and drink intake during the
race were recorded and carbohydrate and energy intake were calculated. Results: Observed glucose
levels ranged between 61.9–252.0 mg/dL. Average glucose concentration differed from the start to the
end of the race (104 ± 15.0 to 164 ± 30.5 SD mg/dL). The total amount of carbohydrate intake during the
race ranged from 0.27 to 1.14 g/kg/h. Glucose concentration positively correlated with running speeds
in segments (P < 0.005). Energy and carbohydrate intake positively correlated with overall running
speed (P < 0.01). Conclusion: The present study demonstrates that continuous glucose monitoring
could be practical to guarantee optimal carbohydrate intake for each ultramarathon runner.
Keywords: sports nutrition; continuous glucose monitoring; carbohydrate; trail running; Freestyle Libre
1. Introduction
For the first time in human history, in 2019, Eliud Kipchoge ran the marathon distance in under two
hours. Recent advances in the area of sports science significantly contributed to his success. In terms
of exercise nutrition, it has been recommended to consume 90 g/h of carbohydrates for endurance
exercise [1,2]. This amount has been suggested based on the maximum oxidation of carbohydrate
as an energy substrate [3,4] and it is noted that the rate-limiting step to oxidizing this amount of
carbohydrate is the gastrointestinal absorption process [1].
A longer distance marathon is known as an ultramarathon, and the popularity of these events has
increased in recent years [5]. The total energy expenditure of a 160 km ultramarathon reaches about
13,000 kcal [6]. Thus, nutritional strategies have to be considered for ultramarathon runners wanting
to improve their race results, but also for those focusing primarily on finishing the event.
GI distress, which is frequently experienced by runners during all types of endurance exercise,
makes the current carbohydrate intake recommendation difficult to achieve [7–10]. Several observation
studies have shown that carbohydrate intake during ultramarathon races is lower than the current
35
Nutrients 2020, 12, 1121
recommendation for carbohydrate intake. In addition to these statements and recommendations, the
optimal nutritional strategies for ultramarathons have been proposed based on a baseline metabolic
model [11]. It has been reported that only one study [12] achieved the carbohydrate amount
suggested in the current recommendation, while others achieved less than the 60 g/h lower level of the
recommendation. The lowest observed average was 31 g/h in slower runners [13].
A recently published position statement of the International Society of Sports Nutrition
recommended the consumption of 150–400 kcal/h (carbohydrate, 30–50 g/h) [9]. Recent practical
recommendations for ultramarathon events offered advice to consume tolerable carbohydrate intake
quantities during exercise, which corresponded to 0.8–1.0 g/kg/h of carbohydrate [14]. These values
were provided by comparing the race diet between fast and slow runners [13] or by comparing the
carbohydrate intake of finishers and non-finishers [12].
Optimal nutrition results in a decreased risk of energy depletion, better performance [10],
the prevention of acute cognitive decline, and improved athlete safety on ultramarathon courses with
technical terrain or those requiring navigation [9]. However, it may prove difficult for the runner to
execute the precise nutrition plan [11] and the carbohydrate requirement for ultramarathon racing
varies greatly depending on the individual [9].
The aim of this study was to evaluate the feasibility of continuous glucose monitoring to improve
the carbohydrate intake of ultrarunners using a continuous glucose monitoring system [15,16].
2.1. Study Design

This observational study was designed to determine the minimum carbohydrate requirement to
maintain blood glucose level and race speed during ultramarathons. All procedures were approved
by the Ryukoku University Human Research Ethics Review Board (No. 2016-08-02). All research
procedures complied with the code of ethics of the World Medical Association (Declaration of Helsinki).
Written informed consent was obtained from all the participants before the commencement of the study.
2.2. Study Population

Seven runners (4 male and 3 female) without injuries volunteered to participate in the study.
All the runners had completed 2 to 3 races certified by the International Trail Running Association and
the sum of finisher’s points exceeded 12 in the last 3 years, demonstrating their experience in running
Ultramarathons. Participant characteristics are presented in Table 1.
Table 1. Clinical characteristics of male and female subjects.
Male Female P
Age (year) 41.5 ± 6.2 42.6 ± 1.2 0.627
Height (cm) 172.9 ± 2.7 158.0 ± 6.5 0.019
Weight (kg) 66.0 ± 9.3 47.9 ± 3.8 0.036
BMI (kg/m2 ) 22.2 ± 2.8 18.9 ± 0.7 0.116
Lean body mass (kg) 56.3 ± 5.9 40.7 ± 4.3 0.012
Fat mass (kg) 22.2 ± 2.8 18.9 ± 0.7 0.116
Values are means ± SD (male, n = 4; female, n = 3).
2.3. Race Course

The present study was conducted during the 2019 Ultra trail Mt. Fuji (https://www.ultratrailmtfuji.
com/), held during the last week of April, around Mt. Fuji in Japan (ambient temperature range:
2.3–19.9 ◦ C). The distance of the course covered 165 km and the total elevation was 7942 m. The course
included trails, rocks, paths, grasslands, and pavements. The course was divided into 11 segments by
10 timing gates where each runner’s passing time was recorded electronically. Distances between each
36
Nutrients 2020, 12, 1121
timing gate were 15 ± 5.4 SD km and varied from 7 to 28 km. Running time and speed between each
timing gate were obtained from the official race web site. Running time between each timing gate was
1:58 ± 0:48 and 2:18 ± 0:52 h:m for the top 5 male and female finishers, respectively. All the runners
had to run with backpacks to carry necessities, including food, and they could replenish food and fluid
at each timing gate.
2.4. Running Speed Data Collection and Standardization

Running speed between each timing gate and overall running speed were obtained from the
official race web site. The standard running speed of male and female participants (designated
as 100%) for each segment were calculated by averaging the top five male and female finishers,
respectively. The running speed of subjects in each segment was standardized using the following
formula. The standardized running speed exceeds 100% only when running at a pace comparable to
the top 1 and 2 places in each gender:
%Running speed = (The subject’s running speed) / ((Average of top 5 finishers’

(1)
running speed in each gender)) × 100,
2.5. Glucose Data Collection and Standardization

Blood glucose profile was monitored by a minimally invasive method known as flash glucose
monitoring (FGM). Its details have been reported elsewhere [15,17,18]. Briefly, the FGM system
(FreeStyle Libre; Abbott Diabetes Care, Alameda, CA) mechanically reads and continuously measures
glucose concentration in the interstitial fluid collected from cells immediately below the skin and
produces the corresponding ambulatory glucose profile. Subjects were asked to attach the device more
than 1 day before the race. The FGM sensor was applied at the back of the upper arm and glucose
concentrations were obtained every 15 min [17].
The glucose concentration of each runner during the race was standardized by subtracting the
resting fasting glucose concentration of the runner and was expressed as an increase from resting
fasting glucose level (Δglucose). The average, highest, lowest, and the difference between the highest
and lowest levels of Δglucose in each segment were used as representative values in each segment
(Figure 1).
Figure 1. Schematic presentation of the standardization of glucose levels during the race. The overall
race course was divided into 11 segments (arrows) by 10 timing gates. The altitude profile of the race
course (filled area) and the change of glucose level (solid line) of the first 12 h of the race is shown as a
representative result. ΔGlucose level was obtained by subtracting the resting fasting glucose concentration
of each runner (dashed line). *, highest value of Δglucose in each segment; †, lowest value of Δglucose in
each segment; dotted line, average value of Δglucose in each segment. Running speed (%) was calculated
by dividing each runner’s running speed by the average running speed of top 5 finishers.
37
Nutrients 2020, 12, 1121
2.6. Diet Supply Data Collection

Runners were asked to record their entire food and drink intake throughout the race. They reported
the timing and volume of consumed food products and fluids based on pictures taken throughout the
race. Food products and fluids consumed more than 60 min before the race start were not included
in the calculation of nutritional intake. The energy and carbohydrate intake during the race were
calculated based on the nutrition information provided by manufacturers. If data was not available,
intakes were calculated based on the standard tables of food composition in Japan 2015 - (7th revised
edition) [19]. The energy and carbohydrate intake were expressed relative to kg of pre-race body
weight, per hour of running time. All foods were categorized with reference to previous research [20]
as: sports drinks (isotonic and hypertonic formulas), gels, cola, other fluids (all other drinks consumed),
sweets, fruits, bars, noodles, bread, rice products and other solids (all other products consumed).
2.7. Statistics
The data reported in the text, tables, and figures are presented as means and standard deviations,
unless otherwise specified. Data were processed and analyzed in GraphPad Prism for Mac (version 8.3.1,
GraphPad Inc., San Diego, CA, USA). Pearson’s correlation coefficients were used to investigate the
associations between running speed, glucose level, and carbohydrate intake. One-way ANOVA
followed by Tukey’s post-hoc test were used to compare the differences between each runner’s blood
glucose level. Results were considered significant when P < 0.05.
3. Results
3.1. General Results

The running speed of the participants ranged from 3.90 to 7.22 km/h with a standardized running
speed ranging from 49.0% to 90.1%.
3.2. Relationship between Glucose Level and Running Speed

All participants were within the expected normoglycemic range during exercise (72–252 mg/dL)
with the exception of one participant who exhibited a lowest value of 61.9 mg/dL as shown in Table 2.
Carbohydrate mainly supplied total energy intake during the race (77.6 ± 8.58SD% of total energy intake).
Table 2. The total energy and nutrient intake, and glucose concentration during the ultramarathon.
Subject FS 1 2 3 MS 4 5 6 7
Sex F F F M M M M M
Running speed (%) 100 89.5 87.9 72.9 100 90.1 70.0 62.0 49.0
(min/km) 6.37 5.70 5.60 4.64 8.01 7.22 5.60 4.96 3.90
Energy intake (kcal/kg/h) - 5.40 4.79 1.91 - 4.37 3.03 1.41 1.46
Carbohydrate intake (g/kg/h) - 1.14 1.04 0.34 - 0.85 0.64 0.27 0.28
Protein intake (g/kg/h) - 0.132 0.061 0.042 - 0.143 0.051 0.021 0.021
Fat intake (g/kg/h) - 0.037 0.040 0.046 - 0.040 0.036 0.030 0.029
Glucose (mg/dL)
During race Average - 131 137 104 - 145 134 121 164
SD - 11.9 30.2 15.0 - 20.4 20.2 22.9 30.5
Highest - 173 224 151 - 193 198 189 240
Lowest - 105 79 62 - 100 94 83 103
Resting fasting - 53 58 40 - 57 68 83 98
FS and MS, female and male standard running speed, which correspond to the average of the top 5 finishers in each
sex. F, female; M, male.
Each runner consumed carbohydrates from liquids, gels, fruits, sweets or solids as shown in Table 3.
Six of 7 runners consumed more than 55% of their carbohydrates from liquids and gels (55.3% to 74.8%)
except for one runner (28.4%, subject 3). Carbohydrate intake from solids ranged from 21.1% to 42.8% in the
six runners and 63.8% in the other runner, who showed the highest fat intake among 7 runners (subject 3).
38
Nutrients 2020, 12, 1121
Table 3. Carbohydrates consumed per product type (g/kg/h).
Subject 1 2 3 4 5 6 7 % of Total
Liquids and gels 0.85 0.71 0.10 0.57 0.35 0.16 0.16 58. 8 ± 15.1
Sports drink 0.34 0.05 0.04 0.01 0.00 0.05 0.04 11.8 ± 10.8
Cola 0.00 0.08 0.00 0.05 0.03 0.06 0.02 6.9 ± 7.5
Gel 0.51 0.57 0.04 0.51 0.32 0.05 0.06 37.2±19.6
Other liquid 0.00 0.01 0.01 0.01 0.00 0.01 0.03 3.0 ± 4.3
Fruits and sweets 0.05 0.06 0.03 0.01 0.06 0.00 0.00 4.2 ± 3.7
Fruit 0.04 0.04 0.02 0.01 0.06 0.00 0.00 3.8 ± 3.5
Sweet 0.01 0.02 0.00 0.00 0.00 0.00 0.00 0.4 ± 0.7
Solids 0.24 0.27 0.21 0.27 0.23 0.10 0.12 37.0 ± 13.9
Bar 0.05 0.00 0.00 0.05 0.00 0.00 0.00 1.6 ± 2.7
Noodle 0.02 0.02 0.08 0.00 0.00 0.01 0.00 4.2 ± 8.1
Bread 0.00 0.05 0.05 0.01 0.04 0.04 0.03 7.9 ± 6.5
Rice product 0.00 0.19 0.09 0.18 0.15 0.04 0.08 18.9 ± 9.7
Other solid 0.17 0.01 0.00 0.04 0.03 0.01 0.00 4.4 ± 4.9
Total 1.14 1.04 0.34 0.85 0.64 0.27 0.28
Subject numbers are identical to Table 2. The subtotal of each category is shown in bold.
The average, highest, lowest, and the difference between the highest and lowest levels of Δglucose
in 11 segments were subjected to correlation analysis between running speed and blood glucose level.
Figure 2 shows the relationship between glucose level and running speeds in each segment. The lowest
(r2 = 0.2397, P = 0.0028; r2 = 0.1397, P = 0.0501 for male and female, respectively) and average (r2 = 0.1650,
P = 0.0155; r2 = 0.0531, P = 0.2381 for male and female, respectively) levels of Δglucose had a significant
positive correlation with running speed, but not for the highest levels of Δglucose (r2 = 0.0005, P = 0.8952;
r2 = 0.0125, P = 0.5704 for male and female, respectively) in male runners. Similar but not significant
tendencies were observed in female runners. Interestingly, a significant inverse correlation (r2 = 0.1198,
P = 0.0417; r2 = 0.0107, P = 0.6011 for male and female, respectively) was observed between running
speed and the difference between highest and lowest (D) in male runners.
$ %

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Figure 2. Scatter plots showing relationships between glucose level and running speed. The lowest
(A), average (B), highest (C), and difference between highest and lowest (D) value of Δglucose levels
were calculated as described in Figure 1. Each plot indicates one segment.
39
Nutrients 2020, 12, 1121
3.3. Relationship between Energy and Carbohydrate Intake and Running Speed
Energy intake exhibited a significant positive correlation with running speed (r2 = 0.8142,
P = 0.0054). Energy intake ranged from 1.41 to 5.40 kcal/kg/h, which is the equivalent of 86.2 to 226.7
kcal/h. A significant correlation was also found between carbohydrate intake and running speed
(r2 = 0.7955, P = 0.0070). Carbohydrate intake ranged from 0.27 to 1.14 g/h/kg (1.1 to 4.6 kcal/h/kg),
which is the equivalent of 16.3 to 52.9 g/h. The energy intake from carbohydrates contributed 63% to
87% of the total energy consumed during the race. No significant correlations were observed between
running speed and energy intake from protein and fat (Figure 3).
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Figure 3. Scatter plots showing relationships between nutrient intake and running speed. The intake
of energy (A), carbohydrate (B), protein (C), and fat (D) were calculated based on consumed food
products and fluids. Each plot indicates one runner.
3.4. Relationship between the Amount of Carbohydrate Intake and Maintenance of Glucose Level during Race
Carbohydrate intake of the seven participants varied within the range of 0.27 to 1.14 g/kg/h
and the carbohydrate intake of four subjects (0.27, 0.28, 0.34, and 0.64 g/kg/h) were less than the
recently published practical recommendations for ultramarathons. The lowest Δglucose levels of the
four subjects were 55.5%, 27.2%, 54.3%, and 66.9% compared to that of the subject who consumed
0.85 g/kg/h, respectively (P < 0.05). Likewise, the average level of Δglucose of the four subjects were
48.2%, 68.6%, and 73.6% compared to that of the subject who consumed 0.85 g/kg/h, respectively (P
< 0.05). Runners who consumed 1.04 or 0.28 g/kg/h of carbohydrate showed higher values in the
highest Δ glucose levels and the difference between the highest and lowest blood glucose among seven
runners, which seemed to be their specific characteristics (P < 0.05, Figure 4).
40
Nutrients 2020, 12, 1121
$ %
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Figure 4. Relationships between carbohydrate intake and the lowest (A), average (B), highest (C),
and the difference between the highest and lowest (D) value of Δglucose levels. Carbohydrate intake
of each runner is expressed in the X-axis. Data are expressed using box-and-whisker plots to indicate
the minimum, first quartile, median, third quartile, and maximum. Bar height indicates the average of
the dots. Values without common superscript are significantly different, P < 0.05
4. Discussion
The aim of this study was to evaluate the feasibility of continuous glucose monitoring to
improve the carbohydrate intake [9,13] of ultrarunners using a continuous glucose monitoring system.
Overall carbohydrate intake in three of seven subjects were far below the recommended carbohydrate
intake (30–50 g/h or 0.8 g/kg/h). A significant positive relationship was observed between higher
carbohydrate intake and faster running speed as was expected from the results of previous studies [12,13].
The present study demonstrates that the avoidance of relatively low blood glucose concentrations,
achieved through the intake of sufficient carbohydrates, impaired running speed during the ultramarathon.
Conversely, there was no association between the highest blood glucose concentrations obtained with
running speed, indicating that control of glucose homeostasis, rather than the rapid availability of
carbohydrates, is the key determinant of performance. Runners consuming less than 0.8 g/kg/h of
carbohydrates tended to have a reduced running speed associated with a result of low blood glucose.
Carbohydrate intake of 30–60 g/h is an established recommendation for endurance sports, with
even higher amounts (i.e., up to 90 g/h and a glucose:fructose ratio of 2:1) being advocated for exercise
bouts lasting more than 3 h [1,2]. However, there is a disparity between this recommendation and actual
intakes in ultramarathon runners. Observation studies have demonstrated that actual carbohydrate
intake during ultramarathons is less than 60 g/h in most runners [6,13,21], including slower runners
consuming 37 g/h [14], with very few runners taking more than 60g of carbohydrates [22,23]. There are
numerous barriers to achieve consumption of 90 g/h of a multiple-transportable carbohydrate blend.
First, the absolute exercise intensity of an ultramarathon is not as high as some other endurance activities
41
Nutrients 2020, 12, 1121
because of its extremely long duration (6, 13, 24, 48, 72 h, 6 or 10 days) [24]. Secondly, the rate-limiting
step for oxidizing 90 g of carbohydrate per hour is intestinal absorption which may be affected by
undertaking exercise of this intensity and duration due to changes in splanchnic blood flow. In addition,
ultramarathon runners lose appetite as a result of heat, endotoxin, or vertical shaking of their digestive
system during rough terrain races [24–26]. Thirdly, a practical limitation is that ultramarathon runners
have to carry their food and fluid in their backpacks during long hours of racing, resulting in an
increase in exercise intensity due to the additional weight being carried [14]. Fourthly, runners may
have physical difficulties in consuming foods when they are keeping balance with both hands when
running down steep mountains or climbing steep slopes.
For these reasons, discrepancies easily occur between the recommended amount and the actual
amount of carbohydrate intake. However, the optimal amount of carbohydrate varies greatly depending
on the individual [9]. Therefore, the application of a continuous glucose monitoring system could be a
practical and fast method to estimate optimal carbohydrate intake for each runner.
Given the duration typical of ultramarathons (6 to 48 h), it is not feasible to meet
carbohydrate consumption in its entirety during a race. Energy deficiency is common in
ultramarathons [8–10,12,13,20,21,27]. Several studies using a doubly labeled water technique or respiratory
gas analysis have estimated that energy expenditure during ultramarathons is about 13000 kcal [6,28,29].
The amount of carbohydrates consumed during a 160 km ultramarathon can be speculated from indirect
calorimetry. The respiratory exchange ratio was 0.91 during the first 64.5km of the 160km race [29]
and was 0.85 immediately after the 330km race [30]. Therefore, carbohydrate oxidation likely provided
50.0%–68.3% of energy expenditure, which is equal to 6500–9100 kcal (1625–2275 g) in the 160 km race.
Gluconeogenesis and hepatic glycogenolysis play an important role to maintain blood glucose
levels during prolonged exercise in a fasted or carbohydrate deficient status. Previous studies have
reported rates of gluconeogenesis and hepatic glycogenolysis as 0.07 g/kg/h and 0.03 g/kg/h, respectively,
in a resting state in low carbohydrate-fed subjects [31]. The sum of these two values (0.1 g/kg/h),
endogenous glucose production, would be the minimum amount of carbohydrate required to maintain
blood glucose during a resting state. The endogenous glucose production significantly increases
to 0.36 g/kg/h during exercise at 55% of peak power output [31] or to 0.48 g/kg/h during exercise
at the lactate threshold level in fasted, well trained subjects [32]. Consistently with these findings,
three subjects in the present study with a carbohydrate intake of less than 0.48 g/kg/h could not
maintain their blood glucose concentrations during the ultramarathon race.
The main limitation of this study is the small number of participants. The present study supports
the effectiveness of a recently published position statement of the International Society of Sports
Nutrition [10] and practical recommendation for ultramarathon participants to prevent hypoglycemia
during exercise. Relationships among carbohydrate intake, the lowest Δglucose, and running speed are
relevant in male runners rather than female runners. These observations coincide with the previously
reported gender-specific differences in fuel utilization during exercise. Women showed higher lipid
oxidation caused by higher plasma adiponectin [33], higher muscle triglyceride utilization [34],
low plasma glucose [35], and higher fasting hepatic glucose uptake [36] compared to men. However,
more subjects are required to conclude that the observed differences between male and female runners
were derived from gender-specific factors.
Hydration and GI distress are negligible factors affecting running speed. Hydration is a factor
causing GI distress [37], but these factors could not be standardized in the study. Dehydration issues
were not observed, which may be associated a steady rain during the race. These two factors should be
quantitatively assessed and statistically analyzed as a factor affecting running speed in larger numbers
of participants.
The insufficient standardization of food intake before and during the race is another limitation.
The following factors should be appropriately controlled in future research: pre-race meals within 48 h
of the start of the race, caffeine intake, gastrointestinal distress, and objective recording of food and
drink intake by action cameras as reported [20].
42
Nutrients 2020, 12, 1121
Another limitation of this study is a slower rise and generally lower glucose peak values in the FGM
system used in the present study as compared with the blood sampling, and this may underestimate
the effect of carbohydrate ingestion on glucose response [18]. Nevertheless, the non-invasive and fast
understanding of fluctuations of glucose level according to the specific characteristics of each athlete
would be useful to plan and modify a personal nutrient strategy during an ultramarathon race.
The other limitation of the present study was large fluctuations in running speed in the
ultramarathon. The running speeds in 11 segments varied in a range of 5.5 to 14.3 km/h and
4.8 to 11.8 km/h even in top five male and female runners, respectively. We speculated that these
fluctuations in running speed were mainly associated with two factors: terrain [26] and physiological
changes such as muscle fatigue and energy deficiency. Therefore, the running speeds of the subjects
were standardized using the top 5 finishers to explore the relationship between blood glucose levels
and running speed. The precise and objective power meters for running, which are already applicable
in cycling studies [38], or accurate physical workload calculation based on GPS monitoring, would
enable more accurate analysis between running performance and blood glucose.
5. Conclusions
In conclusion, the present study demonstrates that continuous glucose monitoring could be
practical to guarantee optimal carbohydrate intake for each ultramarathon runner. Decreases in blood
glucose during ultramarathons may be attributed to many factors, including sub-optimal carbohydrate
intake. Additionally, individual characteristics such as the sex, age, or energy intake of each runner may
have had a greater influence on blood glucose fluctuations across the race; thus, utilizing a continuous
glucose monitor may help inform better race nutrition strategies.
Author Contributions: Conceptualization, K.I.; methodology, K.I., E.M. and T.N.; software, K.I., N.U. and S.K.;
validation, K.I. and T.N.; formal analysis, K.I.; investigation, K.I., N.U., S.K. and T.N.; resources, K.I. and H.O.;
data curation, K.I., N.U., S.K. and T.N; writing—original draft preparation, K.I.; writing—review and editing, K.I.
and T.N.; visualization, K.I. and T.N.; supervision, K.I.; project administration, K.I.; funding acquisition, K.I. and
H.O. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by RYUKOKU University and Nagatasangyo.co. (Hyogo, Japan).
Acknowledgments: We express our gratitude and deep appreciation to Gethin H. Evans, PhD in Manchester
Metropolitan University for supporting academic writing. We thank Masumi Nakao, Yuko Koshiba, Shin Uehira
and Yasuhito Okumura for recruiting participants. We also thank all the participants for their cooperation in
the investigation.
Conflicts of Interest: The author declare that his study has been financed by RYUKOKU university and
Nagatasangyo.co. (Hyogo, Japan). They did not participate in the experimental design, data collection, data
analysis, interpretation of the data, writing of the manuscript, or in the decision to publish the results.
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28. Hill, R.J.; Davies, P.S. Energy expenditure during 2 wk of an ultra-endurance run around Australia. Med. Sci.
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45
nutrients
Communication
Effects of Ashwagandha (Withania somnifera) on
VO2max: A Systematic Review and Meta-Analysis
Jorge Pérez-Gómez 1 , Santos Villafaina 2, *, José Carmelo Adsuar 1 , Eugenio Merellano-Navarro 3
and Daniel Collado-Mateo 4
1 HEME Research Group, Faculty of Sport Sciences, University of Extremadura, 10003 Caceres, Spain;
[email protected] (J.P.-G.); [email protected] (J.C.A.)
2 Physical Activity and Quality of Life Research Group (AFYCAV), Faculty of Sport Science, University of
Extremadura, 10003 Cáceres, Spain
3 Facultad de Educación, Universidad Autónoma de Chile, Talca 3460000, Chile; [email protected]
4 Centre for Sport Studies, Rey Juan Carlos University, Fuenlabrada, 28943 Madrid, Spain;
[email protected]
Received: 15 March 2020; Accepted: 14 April 2020; Published: 17 April 2020
Abstract: The purpose of this study was to systematically review the scientific literature about the
effects of supplementation with Ashwagandha (Withania somnifera) on maximum oxygen consumption
(VO2max ), as well as to provide directions for clinical practice. A systematic search was conducted
in three electronic databases following the Preferred Reporting Items for Systematic Reviews
and Meta-Analyses Guidelines (PRISMA). The inclusion criteria were: (a) VO2max data, with
means ± standard deviation before and after the supplement intervention, (b) the study was
randomized controlled trial (RCT), (c) the article was written in English. The quality of evidence was
evaluated according to the Grading of Recommendations, Assessment, Development and Evaluation
(GRADE) approach. A meta-analysis was performed to determine effect sizes. Five studies were
selected in the systematic review (162 participants) and four were included in the meta-analysis
(142 participants). Results showed a significant enhancement in VO2max in healthy adults and
athletes (p = 0.04). The mean difference was 3.00 (95% CI from 0.18 to 5.82) with high heterogeneity.
In conclusion, Ashwagandha supplementation might improve the VO2max in athlete and non-athlete
people. However, further research is need to confirm this hypothesis since the number of studies is
limited and the heterogeneity was high.
Keywords: ergogenic aids; maximum oxygen consumption; performance sports; physical fitness
1. Introduction
Maximum oxygen consumption (VO2max ) is a physiological parameter that defines the aerobic
capacity of a person. It is an indicator of the cardiorespiratory fitness that describes health status [1] and
sport performance [2]. Focusing on competitive sports, the VO2max , together with running economy
and the anaerobic threshold, is one of the main factors that determine success in endurance activities [3],
and also contributes to increase the team sports performance by increasing work intensity, distance
covered, and number of sprints completed [4]. However, from the point of view of the physical training,
there are still controversies about the best training intensity to enhance the VO2max [5,6].
Apart from sport performance, VO2max has special interest in the field of health. Low values of
VO2max (<17.5 mL·min-1 ·kg-1 ) are associated with an increased risk of mortality and loss of independent
lifestyle in adults and elderly [7], while high values of cardiorespiratory fitness have been associated
with a reduced risk of cardiovascular diseases [8,9]. The VO2max level is also important in children,
where a higher aerobic capacity is related to better quality of life [10].
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Nutrients 2020, 12, 1119
Ashwagandha (Withania somnifera) is a plant in the Solanaceae family. The extract of the
Ashwagandha root has many biological implications due to its diverse phytochemicals [11], so it
has been used, singly or in combination with other natural plants, in many research studies for its
properties: anti-diabetic [12], anti-inflammatory [13], anti-microbial [14], anti-tumor [15], anti-stress [16],
cardioprotective [17], or neuroprotective [18]. It also displays enhanced endothelial function [11],
reduces reactive oxygen species [13], regulates apoptosis [19], and modulates mitochondrial
function [11], showing to be effective to treat aging effects [20], anxiety and stress [21], arthritis [22],
cognitive functions and memory [23], diabetes [12], epilepsy [24], fatigue [25], neurodegenerative
diseases [26], pain [27], thyroid function [28], and skin diseases [29].
In spite of the relevant benefits of supplementation with Ashwagandha, only four meta-analyses
have been carried out evaluating its efficacy on anti-inflammatory effects [30], on impotence and
infertility treatment [31], on neurobehavioral disorders [32] and anxiety [33]. However, there are no
meta-analyses that analyze the effect of Ashwagandha on physical performance. Therefore, the purpose
of this study was to systematically review the scientific literature about the effects of supplementation
with Ashwagandha on VO2max and to provide practical recommendations. Besides, a meta-analysis
was carried out to determine the effect sizes of Ashwagandha on VO2max .
2. Methods
The review was conducted following the statements of the Preferred Reporting Items for Systematic
Reviews and Meta-Analyses Guidelines (PRISMA).
2.1. Literature Search

To find the studies reported in the meta-analysis, several electronic databases were screened:
PubMed (Medline), Web of Science (which includes other databases such as Current Contents Connect,
Derwent Innovations Index, Korean Journal Database, Medline, Russian Science Citation Index, and
Scielo Citation Index) and Google Scholar. The search was conducted in September 2019. The search
terms were: (a) the type of treatment (Ashwagandha or “withania somnifera”) and (b) the outcome
variable (“oxygen consumption” or “aerobic” or “VO2 ”). The search was conducted using the treatment
and the outcome variables, separated by the Boolean operator “and”.
2.2. Study Selection

The inclusion criteria were: (a) VO2max data, with means ± standard deviation (SD) before and
after the supplement intervention; (b) the study was a randomized controlled trial (RCT); (c) the article
was written in English. Two independent authors selected the potentially eligible articles from the
databases. There were no disagreements.
2.3. Quality of the Evidence and Risk of Bias

The quality of the evidence was categorized using the Grading of Recommendations, Assessment,
Development and Evaluation (GRADE) approach. The risk of bias was assessed by the Cochrane
Collaboration’s tool for assessing risk of bias. This tool classified the selection, performance, detection,
attrition, and reporting bias into low, high, or unclear risk of bias.
2.4. Data Collection

Two authors independently extracted data from the studies. The information included: participants,
interventions, comparisons, outcomes, and study design (PICOS), following the recommendations
from the PRISMA statement. Table 1 shows age, sex, sample size, and condition of the participants.
Table 2 presents intervention and the comparison groups, including type of supplementation with the
doses, duration of the study, and the daily frequency of the supplementation. Figure 3 displays results
for the different outcomes. Study design was not included in any table because all studies were RCT.
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Nutrients 2020, 12, 1119
Table 1. Characteristics of the sample.
Groups,
RCT Weeks Age (Years) Country Population
Sample Size and Sex
AS: 20 (M and F)
Shenoy 2012 8 18–27 India Elite cyclists
CG: 20 (M and F)
AS: 16 (M)
Malik 2013 8 16–19 India Hockey players
CG: 16 (M)
AS: 25 (M and F)
Choudhary 2015 12 20–45 India Athletes
CG: 25 (M and F)
AS: 10 (M)
Tripathi 2016 2 18–45 India Healthy adults
CG: 10 (M)
AS: 10 (M and F)
Sandhu 2010 8 18–25 India Healthy adults
CG: 10 (M and F)
RCT: randomized controlled trial; AS: Ashwagandha group; M: males; F: females; CG: control group.
Table 2. Characteristics of the interventions.
Ashwagandha Group Control Group Duration

Dose of the Daily Total
RCT Type of Type of (mg) Study Frequency Dose (g)
Supplementation Supplementation
Ashwagandha in Capsules containing
Shenoy 2012 500 8 weeks twice 56
gelatin capsules starch powder
Sugar power was
Malik 2013 Roots of WS filled in gelatin 500 8 weeks once 28
capsules
Choudhary One capsule of KSM-66 Identical capsules
300 12 weeks twice 50.4
2015 Ashwagandha containing sucrose
WS aqueous extract in
Tripathi 2016 Maize starch capsule 330 2 weeks once 4.62
the capsule form
WS filled in gelatin Capsules filled with
Sandhu 2010 500 8 weeks once 28
capsules flour
RCT: randomized controlled trial; KSM-66: commercial name of an Ashwagandha extract; WS: Withania Somnifera.
Total dose was calculated as: total dose (g) = (dose (mg) × daily frequency × study duration (days))/1000.

The main outcome of this meta-analysis was VO2max . The meta-analysis was conducted using
the Revision Manager (RevMan) software (version 5.3) obtained from Cochrane Collaboration web.
Post-intervention mean and SD were extracted and used for meta-analyses. All articles reported VO2
max as mL/kg/min. Mean difference was calculated using a random model. The heterogeneity between
the studies was calculated using Tau2 , I2 , and Chi2 tests. Although there is no consensus about the
definition of “mild”, “moderate”, or “severe” heterogeneity, Higgins and Thompson [34] suggested
that values for I2 higher than 56% would mean large heterogeneity while values lower than 31% would
be related to low heterogeneity.
3. Results

The PRISMA flow diagram is showed in Figure 1. A total of 129 records were identified, 9 of
which were removed because they were duplicated. Of the remaining 120 articles, 92 were excluded
because they were not related with the topic, 4 studies were not written in English, and 4 were reviews.
After reading the remaining 20 articles, another 15 studies did not meet the inclusion criteria and were
excluded. Therefore, 5 studies were included in the systematic review. However, the article by Sandhu
et al. [35] was excluded from meta-analysis due to the odd results. In this regard, they evaluated
healthy young males and females aged between 18 and 25 with body mass index between 18 and 25.
Their mean peak VO2max was lower than 14mL/kg/min, which is so much lower than expected for
healthy young people and less than half the mean of the rest of the included studies (46.18 mL/kg/min).
49
Nutrients 2020, 12, 1119
We tried to contact with the authors in order to obtain a reason for that, but at the time this article was
considered for publication, we did not receive a response. Considering that in the article authors did
not explain an incremental test to obtain the VO2max , we believe that they measured the gas exchange
at rest, reporting the oxygen consumption (VO2 ). Therefore, this article was included in systematic
review but not in the meta-analysis.
Figure 1. Flow chart delineating the complete systematic review process.
3.2. Quality of Evidence and Risk of Bias

The evidence of the effects on VO2max was initially classified as “high quality” due to all the
selected articles were RCT, but the evidence dropped twice because of the small sample size and due
to the high degree of heterogeneity. Therefore, the final quality of the evidence was low. The Cochrane
Collaboration’s tool for assessing risk of bias (Figure 2) showed that the poorer scores were obtained in
the performance and detection bias due to unclear reporting.
3.3. Study Characteristics

Study characteristics are summarized in Table 1. The total number of participants included in this
systematic review were 162. Of these, 81 belonged to the Ashwagandha group and 81 were the placebo
(control) group. The age ranged from 16 to 45 years old. The sample was comprised exclusively of
healthy adults and athletes.
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Nutrients 2020, 12, 1119
Figure 2. The Cochrane Collaboration’s tool for assessing risk of bias.
3.4. Interventions
The characteristics of the Ashwagandha supplementation and placebo group are displayed in
Table 2. The doses varied from 300 to 500 mg and the daily frequency intake was once or twice a day.
The total duration of the intervention varied from 2 to 12 weeks.

The study of Choudhary et al. [36] found a significant group*treatment interaction in the VO2max .
The remaining four articles only found within-group improvement in VO2max after the supplement
intervention [35,37–39].
Regarding meta-analysis results, a significant (p = 0.04) mean difference was observed. Figure 3
showed a mean difference of 3.00 (95% CI from 0.18 to 5.82). The heterogeneity level was large
according to the I2 = 84%. The quality of the evidence was low according to the GRADE classification.
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Nutrients 2020, 12, 1119
Figure 3. Meta-analysis results of the effects of Ashwagandha supplementation on VO2max .
52
Nutrients 2020, 12, 1119
4. Discussion
The purpose of this study was to systematically review the scientific literature about the effects of
supplementation with Ashwagandha on VO2max and to carry out a meta-analysis to determine the
overall effect. After 20 articles were assessed for eligibility, 15 articles were excluded since they did not
report VO2 max . A total of 5 articles were included in the systematic review [35–39]. However, one article
was excluded from the meta-analysis [35] since the reported mean VO2max was abnormally low for
healthy young people and less than half the mean of the rest of the included studies (46.18 mL/kg/min),
which may indicate that they were not actually reporting VO2max but VO2 at rest. The results of this
meta-analysis showed that supplementation with Ashwagandha may be useful to improve VO2max in
athletes [36,38,39] and healthy adults [37]. Table 2 displayed the amount of Ashwagandha used in
each study, which varied from 330 up to 1000 mg/day, which is inside the limits, 750 to 1250 mg/day,
found to be well tolerated and safe [40]. In this regard, none of the five articles reported any relevant
side effect as a consequence of the treatment, achieving a high compliance with the treatment and very
low number of dropouts.
The two studies that achieved the highest treatment effect and effect size [36,39] were those with
the highest Ashwagandha intake (>50 g in the whole program). Therefore, it seems like the higher the
dose, the higher the improvement in VO2 . However, the study by Tripathi, Shrivastava, Ahmad Mir,
Kumar, Govil, Vahedi, and Bisen [14] did not observe any significant difference between the effects of a
330 mg intake and the effects of a 500 mg intake after 2 weeks. Therefore, further studies comparing
the effect of different doses, as well as studies with longer duration are needed.
In general terms, the overall effects were better in those studies with a sample comprised of
athletes [36,38,39] compared with the studies with healthy adults [14,39]. This is interesting since, as
expected, baseline levels were higher in athletes and, consequently, larger improvements were expected
in non-athlete healthy adults. It could be that the effects of supplementation with Ashwagandha might
be linked to the physical activity levels of the participants, promoting and increasing the physiological
adaptations to physical exercise. However, this hypothesis should be explored in future studies.
The VO2max defines the body’s ability to transport and utilize oxygen, so this physiological parameter
is associated with endurance performance. Many factors contribute to the VO2max values, including
genetic predisposition [41], enzymes [42], muscle fiber type [43], or training [44]. It is also known that
nutritional supplementation can improve the effects of training and reach higher performance [45].
Previous studies with Ashwagandha administration observed improvement in working capacity test
in rats by increasing the swimming endurance test [46]. As endurance performance is determined
by mitochondrial function, some reasons for the Ashwagandha to improve cardiorespiratory fitness
can be the significant effects observed on mitochondrial and energy levels, by reducing the succinate
dehydrogenase enzyme activity in the mitochondria and benefiting Mg-ATPase activity [47]. Previous
studies showed that Ashwagandha significantly enhanced the hemoglobin concentration and red blood
cells in animals [48] and also in humans [38], with the subsequent increase in the capacity to transport
oxygen to the muscles. Moreover, it should be considered that Ashwagandha has shown to have
anti-fatigue [49,50] and anti-stress [51] actions. This could be connected to the significant improvement
in the time to exhaustion of the experimental group that could be observed in the study of Shenoy,
Chaskar, Sandhu, and Paadhi [39]. Some of the chemical constituents of Whitania somnifera [52] such as
flavonoids, alkaloids, and steroidal lactones (withanolides) or the antioxidants (superoxide dismutase,
catalase, and glutathione peroxidase) could be behind the improvements of VO2max . Therefore, further
studies are needed to explore which are the chemical constituents and mechanism that may explain
the potential improvement in the VO2max .
Although all mechanisms by which Ashwagandha can improve the VO2max have not been
described yet and future studies are needed to elucidate that improvement, it is known that
Ashwagandha exhibits little or no associated toxicity [53], so it seems that this Ayurvedic herb
“Ashwagandha” (Withania somnifera) can be safely used for improving cardiovascular fitness in healthy
53
Nutrients 2020, 12, 1119
adults and also in athletes, offering an additional alternative as a nutritional supplement to enhance
VO2max.
Some limitations in the present meta-analysis can be mentioned. The first one is related to the
search strategy, only articles published in English were included and a few databases were used.
Another limitation can be the large heterogeneity in the included articles. Different doses, levels of
physical activity, or the inclusion of both women and men in the protocols make it very difficult
to achieve a high level of evidence. In addition, the systematic review and meta-analysis was not
prospectively registered in any public database. Furthermore, in order to have a better understanding
of long-term ergogenic benefit and potential side effects from Ashwagandha root extract, longer
duration studies are needed.
5. Conclusions
Ashwagandha supplementation might improve the VO2max in athlete and non-athlete people.
The analyzed studies used oral administration of Ashwagandha which varied between 2 and 12
weeks with intakes between 300 to 1000 mg/day. Due to the limited number of studies included in
this systematic review and meta-analysis, further research is needed to confirm the effects and the
recommended dose.
Author Contributions: Conceptualization, J.P.-G., J.C.A. and D.C.-M.; methodology, J.P.-G., S.V., E.M.-N. and
D.C.-M.; software, J.C.A., and D.C.-M.; formal analysis, J.P.-G., S.V., J.C.A., E.M.-N., and D.C.-M.; investigation,
J.P.-G., S.V., J.C.A., E.M.-N., and D.C.-M.; data curation, J.P.-G., J.C.A. and S.V.; writing—original draft preparation,
J.P.-G., S.V. and D.C.-M.; writing—review and editing, J.P.-G., S.V., J.C.A., E.M.-N. and D.C.-M.; supervision, J.P.-G.,
S.V., J.C.A., E.M.-N. and D.C.-M. All authors have read and agreed to the published version of the manuscript.
Funding: S.V. is supported by a grant from the regional Department of Economy and Infrastructure of the
Government of Extremadura and European Social Fund (PD16008).
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57
nutrients
Article
Exogenous Ketone Supplements Improved Motor
Performance in Preclinical Rodent Models
Csilla Ari 1,2, *, Cem Murdun 3 , Craig Goldhagen 3 , Andrew P. Koutnik 3,4 , Sahil R. Bharwani 1 ,
David M. Diamond 1,3 , Mark Kindy 5,6,7 , Dominic P. D’Agostino 2,3,4 and Zsolt Kovacs 8
1 Department of Psychology, Behavioral Neuroscience Research Laboratory, University of South Florida,
Tampa, FL 33620, USA; [email protected] (S.R.B.); [email protected] (D.M.D.)
2 Ketone Technologies, Tampa, FL 33612, USA; [email protected]
3 Department of Molecular Pharmacology and Physiology, Laboratory of Metabolic Medicine, Morsani
College of Medicine, University of South Florida, Tampa, FL 33612, USA; [email protected] (C.M.);
[email protected] (C.G.); [email protected] (A.P.K.)
4 Institute for Human and Machine Cognition, Ocala, FL 34471, USA
5 Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL 33612,
USA; [email protected]
6 James A. Haley VA Medical Center, Tampa, FL 33612, USA
7 Shriners Hospital for Children, Tampa, FL 33612, USA
8 Savaria Department of Biology, ELTE Eötvös Loránd University, Savaria University Centre,
Károlyi Gáspár tér 4., 9700 Szombathely, Hungary; [email protected]
* Correspondence: [email protected] or [email protected]; Tel.: +1-813-240-9925
Received: 3 July 2020; Accepted: 13 August 2020; Published: 15 August 2020
Abstract: Nutritional ketosis has been proven effective for neurometabolic conditions and disorders
linked to metabolic dysregulation. While inducing nutritional ketosis, ketogenic diet (KD) can
improve motor performance in the context of certain disease states, but it is unknown whether
exogenous ketone supplements—alternatives to KDs—may have similar effects. Therefore, we
investigated the effect of ketone supplements on motor performance, using accelerating rotarod test
and on postexercise blood glucose and R-beta-hydroxybutyrate (R-βHB) levels in rodent models with
and without pathology. The effect of KD, butanediol (BD), ketone-ester (KE), ketone-salt (KS), and
their combination (KE + KS: KEKS) or mixtures with medium chain triglyceride (MCT) (KE + MCT:
KEMCT; KS + MCT: KSMCT) was tested in Sprague-Dawley (SPD) and WAG/Rij (WR) rats and in
GLUT-1 Deficiency Syndrome (G1D) mice. Motor performance was enhanced by KEMCT acutely, KE
and KS subchronically in SPD rats, by KEKS and KEMCT groups in WR rats, and by KE chronically in
G1D mice. We demonstrated that exogenous ketone supplementation improved motor performance
to various degrees in rodent models, while effectively elevated R-βHB and in some cases offsets
postexercise blood glucose elevations. Our results suggest that improvement of motor performance
varies depending on the strain of rodents, specific ketone formulation, age, and exposure frequency.
Keywords: ketone ester; ketogenic diet; ketone salt; MCT; rotarod; R-βHB
1. Introduction
Motor impairment can be caused by injury or degeneration to the motor cortex, premotor cortex,
motor tracts, or associated pathways in the cerebrum, cerebellum, or neuromuscular junction. These
pathological changes have been observed in a variety of neurological conditions, such as Alzheimer’s
Disease (AD), Huntington’s Disease (HD), Parkinson’s Disease (PD), Amyotrophic Lateral Sclerosis
(ALS), Glucose 1 Deficiency Syndrome (G1D), or those who have suffered a cerebral vascular accident
(CVA) stroke, and in patients with traumatic brain injuries (TBI) [1–6]. These neurological conditions
present with metabolic impairment, neuroinflammation, and other related molecular characteristics.
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Nutritional ketosis significantly improved outcomes and motor function in HD, PD, ALS, G1D, and AD
through various mechanisms, such as reduced proinflammatory cytokines, decreased mitochondrial
damage, and preservation of cellular bioenergetics. These results suggest that the mechanism of action
for ketone bodies to enhance/preserve motor performance is multifactorial, but has significant overlap
between biological pathways [1–9]. However, the predominant mechanism of action of nutritional
ketosis on motor performance is largely unknown, as is the optimal method of administration.
The ketogenic diet (KD) was designed to induce nutritional ketosis, initially employed for its
metabolic benefits and ability to treat epilepsy [10]. Physiologically, the metabolic response to a KD
closely resembles the insulin suppression associated with fasting if total calories and ketogenic ratios
(3:1 to 4:1 ratio, by weight, of fat to a combination of protein and carbohydrates) are maintained [5].
Despite the efficacy of the KD clinically, patient compliance can be low due to the strict nutritional
requirements, fat intolerance, or potential complications and side effects [11,12]. Subjects report
difficulty maintaining ketosis as excess consumption of carbohydrates or protein can rapidly shift
the body back towards glycolysis and inhibit ketogenesis [13]. Alternative solutions to achieving
nutritional ketosis are needed, and exogenous ketone supplementation has been reported to be an
effective alternative to KD in the context of various disease states [11,14–19].
Under normal physiological conditions and when adhering to a “traditional” western diet
typically rich in carbohydrate, glucose is the primary metabolic fuel in the CNS and skeletal muscle [20].
However, during low glucose bioavailability and hepatic glycogen depletion, free fatty acids are
mobilized from adipose tissue and released into the bloodstream to be used directly as fuel or converted
to ketone bodies in the liver through the process of ketogenesis to sustain metabolic demands [21].
The primary ketone bodies produced, acetoacetate (AcAc) and R-β-hydroxybutyrate (R-βHB), are
subsequently released into the bloodstream and serve as an alternative fuel for most tissues, but
especially the central nervous tissue, as long-chain fatty acids cannot readily be used by the brain [22].
Hyperketonemia is the metabolic state characterized by an elevation of blood ketone bodies and
is observed in individuals who adhere to a KD or are fasted for extended periods of time [22]. In this
state, a large portion of tissue energetic requirements are met via fatty acid oxidation and ketolysis [23].
Under normal physiological conditions, blood ketone levels rarely exceed 0.01 mmol/L and account for
less than 3% of total brain metabolism [24]. However, in periods of nutritional ketosis, characterized
by blood ketone levels of 0.5–6 mmol/L, ketone bodies cross the blood–brain barrier (BBB) through
monocarboxylate class transporters [25] and serve as supplemental fuel accounting for up to 60%
of brain energy metabolism [21,26]. Once inside of the cell’s mitochondria, the ketone bodies are
converted into Acetyl CoA, enter the TCA cycle, and generate the reduced intermediates (NADH and
FADH2 ) needed to sustain ATP production [22].
Supplementation with ketone (ketogenic) supplements, such as ketone ester (KE), has proven
effective in achieving a state of nutritional ketosis independent of carbohydrate restriction [14,17,18,27,28].
Ketone supplementation that contains either KE, ketone salt (KS), medium chain triglycerides (MCTs),
or their combinations (e.g., KEKS, KEMCT, and KSMCT) has been studied in both animal models and
humans. It has been demonstrated that exogenous ketone supplementation is a safe and effective
option to induce nutritional ketosis [16,29–32], and, consequently, evoke alleviating effects of ketosis
on motor performance similar to KD [2,4,6,33]. Indeed, it was suggested that administration of KE and
MCTs may evoke beneficial effects on motor dysfunction [34,35]. However, it is unknown whether
different exogenous ketone supplements and their combinations would increase motor performance in
animal models under different conditions associated with motor function impairment. Our previous
study also demonstrated that exogenous ketones have a blood glucose lowering effect [27]; however,
the hormonal response to exercise can cause stored glucose to be released from the liver and skeletal
muscle while temporarily raising blood glucose levels. Thus, the main goal of the present study was to
determine if specific ketogenic supplements and/or combinations would improve motor performance
in rodents with pathology (Wistar Albino Glaxo/Rijswijk, WAG/Rij/WR rats and Glut1 Deficiency
Syndrome/G1D mice) and without pathology (Sprague-Dawley/SPD rats) [36,37] and to test whether
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Nutrients 2020, 12, 2459
they are able to offset the postexercise induced blood glucose elevation. WR rat strain is an accepted
model of absence epilepsy with comorbidity of low-grade depression, but without pathological changes
in locomotor activity/motor performance [38–40], while G1D mice are models of Glut1 Deficiency
Syndrome and show motor dysfunction [37,41]. We hypothesized that nutritional ketosis induced by
various ketone therapeutics (KD and exogenous ketone supplements) can improve motor performance
in rodent models, as defined by latency to fall from the accelerated rotarod and can offset blood glucose
elevation in postexercised states.
2.1. Animals
Three rodent models were used for the experiments: SPD rats (male, 4 months old and 1 year old,
320–360 g and 540–660 g, respectively, Harlan Laboratories), WR rats (male, 6 months old, 320–360 g,
breeding colony, Eötvös Loránd University, Savaria University Centre, Szombathely, Hungary), and
G1D mice (male, 3–5 months old, 17–27 g, breeding colony, University of South Florida, Morsani
College of Medicine, Tampa, FL, USA) that were housed at either the College of Medicine Animal
Facility (Morsani College of Medicine, University of South Florida, Tampa, FL, USA) or at the Savaria
Department of Zoology (Eötvös Loránd University, Savaria University Centre, Szombathely, Hungary).
Standard laboratory conditions (12:12 h light-dark cycle) were maintained for the animals that were
housed in air-conditioned rooms at 22 ± 2 ◦ C in groups of 2–4.
Institutional Animal Care and Use Committee (IACUC; Protocol #0006R) of the University of
South Florida (University of South Florida, Tampa, FL, USA) and Hungarian Act of Animal Care and
Experimentation (1998. XXVIII. Section 243/1998) and the regulations for animal experimentation in
the European Communities Council Directive of 24 November 1986 (86/609/EEC) guidelines were
followed during the experimental procedures. Experiments were approved by the Animal Care and
Experimentation Committee of the Eötvös Loránd University (Savaria University Centre) and by the
National Scientific Ethical Committee on Animal Experimentation (Hungary) under license number
VA/ÉBNTF02/85–8/2016. We made all efforts to reduce the number of animals used.
2.2. Ketogenic Compounds

Ad libitum access to water and standard rodent chow (standard diet: SD, NIH-31 Rodent
Chow; Envigo), or ketogenic rodent food (KD; TD. 10911; Envigo), or SD with ketone supplements
(Table 1, and Supplementary Tables S1 and S2) were provided for the animals. The KD used in this
study was modified from TD.10787 (Teklad) to remove maltodextrin to make the diet essentially
free of carbohydrate. The KD had a 2:1 ratio of n-3 to n-6 fatty acids and a 1.5:1 ratio of fat to
protein + carbohydrate. Compared to TD.10787 (common KD formula), TD.10911 had the same %
fat (wt/wt), but it had a higher % kcal from fat, because maltodextrin was removed and replaced
with cellulose. KE (R,S 1,3-butanediol-acetoacetate diester) was synthesized by D’Agostino, 2013, as
previously described [17]. The KS (Na+ /K+ – R,S βHB mineral salt) was mixed into a 50% solution
supplying approximately 375 mg/g of pure βHB and 125 mg/g of Na+ /K+ in a 1:1 ratio. KE and KS
development and synthesis were performed in collaboration with Savind Inc. Human food-grade MCT
oil (~60% caprylic triglyceride/40% capric triglyceride, Now Foods, Bloomingdale, IL, USA) was used
for the experiments. KS and KE were mixed with MCT in a 1:1 ratio, resulting the KSMCT and KEMCT
combinations, respectively. KE was mixed with KS in a 1:1 ratio to make KEKS. R,S-1,3-butanediol
(BD) was purchased from Sigma (Milwaukee, WI, USA).
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Nutrients 2020, 12, 2459
Table 1. Macronutrient information of each diet. (More details about the ingredients of each diet can
be found in Supplementary Tables S1 and S2).
Standard Diet (SD) Ketogenic Diet (KD)

Protein, % of kcal 24 22.4
Carbohydrate, 5 of kcal 62 0.5
Fat, % by kcal 14 77.1
kcal/g 3.0 4.7
2.3. Exposure Schedule

For the trials involving SPD and WR rats, intragastric delivery by oral gavage was used.
To acclimatize the rodents, each animal was orally gavaged with water for five days prior to treatment
(Figure 1). After acclimatization and baseline measurements (5th days), the rats were orally gavaged
either once with different exogenous ketone supplements (acute treatment on the 6th day; 5 g/kg for
SPD rats) and the effects (motor function and blood measurements) were measured after 30 min−1 h, or
they were gavaged once daily for 7 days (subchronic treatment between 6th and 12th day; 5 g/kg/day
for SPD rats and 2.5 g/kg/day for WR rats) and the effects were recorded after 24 h and after 7 days
(Figure 1). In relation to G1D mice with chronic exposure, ketone supplements were administered
daily for 10 weeks and the effects were measured after 3, 6, and 10 weeks treatment (Figure 1).
Figure 1. Experimental design for the different exposure schedules (acute, subchronic, and chronic).
BL: Baseline measurement; blood: blood draw with glucose and R-βHB measurements; RR: Rotarod
test; blood: blood draw with glucose and R-βHB measurements.
2.4. Treatment Groups

One-year-old SPD rats were divided into 6 groups for the acute treatment and were fed with
SD and they were administered a single oral gavage. The treatment groups included water (control,
n = 10), BD (n = 8), KE (n = 12), KSMCT (n = 8), KEKS (n = 12), and KEMCT (n = 8). Motor performance
62
Nutrients 2020, 12, 2459
was evaluated prior to the beginning of dietary treatment (baseline; 5th day of habituation) and 30 min
after gavage.
Subchronic experiments used 4-month old SPD rats that were divided into 5 groups and fed with
either SD, KD and supplemented with a once-daily oral gavage for 7 days. Treatment groups included
control (SD, water gavage, n = 11), KD (n = 10), KE (n = 9), KS (n = 9), and KSMCT (n = 10). The animals
were evaluated for motor performance prior to the beginning of dietary treatment (baseline), 24 h after
1st treatment (KD or gavage), and 1 h after the 7th treatment.
The WR rats with subchronic exposure were divided into 6 groups. They were fed with SD and
orally gavaged daily with either water (SD, control; n = 9) or KE (n = 9), KS (n = 9), KSMCT (n = 9),
KEKS (n = 9), KEMCT (n = 9) for 7 days. Rotarod test was carried out similarly to subchronically
treated SPD rats.
The G1D mice exposed to chronic treatment were split into four groups, SD (control; n = 12),
KD (n = 12), or the SD supplemented with KS (n = 12) or KE (n = 12) and were treated for 10 weeks.
Rotarod test was performed before the beginning of dietary treatment (baseline) and after 3, 6, and
10 weeks of treatment.
2.5. Motor Performance Testing

Motor performance was evaluated using the accelerating rotarod test on a RotaRod Rotamex 5
(rotarod; Columbus Inst., Columbus, OH, USA). The rats were placed on the rods of the accelerating
rotarod, and the time the animals remained on the rod was measured. The rotarod was set to accelerate
from zero to 40 rpm in 180 s. To acclimate the animals to both the equipment and the task, prior to the
administration of a test, animals were trained on the rotarod for 5 consecutive days. Thus, habituation
to rotarod test was parallel with habituation to oral gavage (Figure 1). Each day of training consisted of
3 sessions, with a 120 s rest period between trials. Animals were placed on the rotarod and timed until
they fell from the rotating rod. Following last trial, the blood measurements were collected within
10 min.
2.6. Blood Glucose and R-βHB

Whole blood (~10 μL) was acquired from saphenous vein (rats) and tail vein (mice) for analysis of
blood glucose (mg/dL) and R-βHB (mmol/L) with Precision XtraTM (Abbott Laboratories, Abbott Park,
IL, USA). Note that the Precision XtraTM measures R-βHB exclusively—not S-βHB, AcAc, or
Acetone—therefore, total ketone level may be somewhat underrepresented. R/D and S/L are two
stereoisomers, called enantiomers, that are mirror images of each other. While R-βHB is the normal
product of human and mouse metabolism, it is metabolized much faster than S-βHB is. S-βHB is
not a normal product of human metabolism, however, it is a transient intermediate of β-oxidation
of fatty acids, therefore, administration of the same amount of S-βHB may lead to higher level and
more sustained blood levels of S-βHB, compared to similar administration of R-βHB [42]. Blood was
drawn prior to the beginning of the treatments (5th day of habituation), and this value was used as the
established baseline. Treatment-generated changes in glucose and R-βHB were measured (60 min)
after the beginning of treatment (acute treatments, SPD rats). Blood was drawn after treatment started,
at either 1 h, 24 h, or after 7 daily treatments (subchronic treatments, SPD, and/or WR rats). During
chronic treatments (G1D mice), blood was drawn before treatment started (baseline) and at week 3, 6,
and 10 after beginning treatment.
2.7. Statistics
Data are presented as the mean ± standard error of the mean (SEM). The effects of ketogenic
agents on both R-βHB and glucose, as well as motor performance (latency to fall: rotarod) were
compared to experimental controls and respective baseline levels. GraphPad Prism (6.0a) was used
for data analysis. Comparisons of data were made using a one or two-way ANOVA with Tukey’s
multiple comparisons. Results were considered significant when p values were less than 0.05. Results
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Nutrients 2020, 12, 2459
are indicated on figures using the following notations *: p < 0.05, **: p < 0.01, ***: p < 0.001, and
****: p < 0.0001 level of significance.
3. Results
3.1. Changes in Motor Performance, Blood Glucose, and R-βHB Levels in SPD Rats with Acute Exposure
In acutely exposed one-year-old SPD rats, the latency to fall from an accelerating rotarod was
significantly elevated in KEMCT group, compared to baseline (p = 0.0011, Figure 2A). The KEKS group
showed a trend of increase, however, the results were nonsignificant, while the BD, KSMCT, and
KE groups also showed no significant change in motor performance. The percent change in latency
to fall was significantly higher in KEMCT group, compared to control (p = 0.003, Figure 2B). Blood
R-βHB levels in postexercise state were elevated significantly in KSMCT group, compared to baseline
(p = 0.0099) and control (p = 0.0040), in KEKS group, compared to baseline (p = 0.0021) and control
(p = 0.0119), in KEMCT group, compared to baseline (p < 0.0001) and control (p < 0.0001), and in KE
group, compared to baseline (p < 0.0001) and control (p < 0.0001, Figure 2C). The BD group displayed a
nonsignificant increase in blood R-βHB levels. The blood glucose levels in postexercise state were
elevated significantly after 1 h in the control group (p = 0.0064), in BD group (p < 0.0001), in KSMCT
group (p < 0.0001), in KEKS group (p = 0.0048), and KE group (p = 0.0255), compared to their respective
baselines (Figure 2D).
Figure 2. Changes in motor performance and blood parameters from postexercise state for one-year old
Sprague-Dawley rats, 1 h following treatment. (A) Latency to fall (sec) on accelerating rotarod at each
timepoint. (B) Percent change in latency to fall on accelerating rotarod at 1 h, compared to baseline.
(C) Blood R-βHB levels in postexercise state at each timepoint. (D) Blood glucose levels in postexercise
state at each timepoint. Abbreviations: B: Baseline; 1 h: 1 h timepoint; R-βHB: R-beta-hydroxybutyrate;
BD: butanediol; KE: ketone ester; KEKS: combination of ketone ester and ketone salt (KS); KEMCT:
ketone ester in combination with medium chain triglyceride (MCT); KSMCT: ketone-salt in combination
with medium chain triglyceride (MCT); SPD: Sprague-Dawley rats. *: p < 0.05, **: p < 0.01, and
****: p < 0.0001 level of significance.
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Nutrients 2020, 12, 2459
3.2. Changes in Motor Performance, Blood Glucose, and R-βHB Levels in SPD Rats with Subchronic Exposure
In the 4-month-old SPD rats after subchronic treatment the latency to fall from the accelerating
rotarod was significantly decreased for the KD group at 24 h (p = 0.0296) and at 7 days (p = 0.0133),
compared to baseline (Figure 3A). The latency to fall increased in KE group at 24 h, compared to
baseline (p = 0.0313), and at 7 days, compared to the control (p = 0.0413) and baseline (p = 0.0106).
The latency to fall increased in KS group at 24 h, compared to the control (p = 0.0138) and baseline
(p = 0.039), and at 7 days, compared to the control (p = 0.011). The KSMCT group displayed no
significant change in latency to fall. No groups had significantly different percent change in latency to
fall, compared to baseline or control (Figure 3B). Blood R-βHB levels increased significantly in the KD
group at 24 h, compared to baseline (p = 0.038) and control (p < 0.0001) and in the KE group at 24 h,
compared to control (p = 0.0325, Figure 3C). After 7 days the blood R-βHB levels increased significantly
in KD and KS groups, compared to the control (p < 0.0001 and p = 0.0194 respectively), and in KSMCT,
compared to baseline (p < 0.0001), 24 h (p < 0.0001), and control (p < 0.0001). Blood glucose level at
24 h was significantly decreased in KD group, compared to control (p < 0.0001), and in the KE group,
compared to control (p < 0.0001) and baseline (p = 0.0047, Figure 3D). After 7 days of treatment, the
postexercise blood glucose level was decreased in KSMCT group, compared to baseline (p < 0.0001),
24 h (p < 0.0001), and control (p < 0.0001). An increase in blood glucose level was observed in the KE
group relative to its 24-h reading (p = 0.0049).
Figure 3. Motor performance and blood parameters in postexercise state for 4-month-old
Sprague-Dawley rats after subchronic treatment, measured at baseline, 24 h and at 7 days following
treatment. (A) The latency to fall (sec) on the accelerating rotarod was presented at each timepoint.
(B) The percent change in latency to fall on the accelerating rotarod presented at 24 h and at 7 days,
compared to baseline. (C) Blood R-βHB levels are presented in postexercise state at each timepoint.
(D) Blood glucose levels are presented in postexercise state at each timepoint. Abbreviations: B: Baseline;
24 h: 24 h timepoint; 7d: 7 days timepoint; R-βHB: R-beta-hydroxybutyrate; KD: ketogenic diet; KE:
ketone ester; KS: ketone salt; KSMCT: ketone salt in combination with medium chain triglyceride
(MCT); SPD: Sprague-Dawley rats. *: p < 0.05, **: p < 0.01, and ****: p < 0.0001 level of significance.
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3.3. Changes in Motor Performance, Blood Glucose, and R-βHB Levels in WR Rats with Acute and
Subchronic Exposure
In WR rats with acute and subchronic exposure, the latency to fall on accelerating rotarod was
significantly decreased in KSMCT group at 1 h and at 7 days, compared to the baseline (p < 0.0129;
p = 0.0078; respectively, Figure 4A). In KEKS group the latency to fall increased at 1 h, compared
to baseline (p < 0.0001) and control (p < 0.0001), and at 7 days, compared to baseline (p < 0.0001),
1 h (p < 0.0001), and control (p < 0.0001). In the KS group the latency to fall significantly decreased
between 1 h and 7 days (p = 0.0138). The latency to fall increased in KEMCT group at 1 h, compared to
baseline (p < 0.0001) and control (p < 0.0001), and at 7 days, compared to baseline (p < 0.0001) and
control (p < 0.0001). The percent change in latency to fall on the accelerated rotarod was significantly
increased in the KEKS group at 1 h, compared to control (p = 0.0001), and at 7 days, compared to control
(p < 0.0001, Figure 4B). The KEMCT group at 1 h had a significantly increased percent change in latency
to fall, compared to control (p < 0.0001) and similarly at 7 days, compared to control (p < 0.0001).
Figure 4. Changes in motor performance and blood parameters in postexercise state for WR rats with
acute and subchronic exposure, at baseline, at 1 h and 7 days following treatment. (A) The latency
to fall (sec) on accelerating rotarod at each timepoint. (B) The percent change in latency to fall on
accelerated rotarod, compared to baseline at 1 h and 7 days post treatment. (C) Blood R-βHB levels are
presented in postexercise state at each timepoint. (D) Blood glucose levels are presented in postexercise
state at each timepoint. Abbreviations: B: Baseline; 1 h: 1 h timepoint; 7d: 7 days timepoint; R-βHB,
R-beta-hydroxybutyrate; KE: ketone ester; KEKS: combination of ketone ester and ketone salt (KS);
KEMCT: ketone ester in combination with medium chain triglyceride (MCT); KS: ketone salt; KSMCT:
ketone-salt in combination with medium chain triglyceride (MCT); WR: WAG/Rij rats. *: p < 0.05,
**: p < 0.01, ***: p < 0.001 and ****: p < 0.0001 level of significance.
Blood R-βHB levels were increased significantly after 1 h in the KE, KSMCT, KEKS, and KEMCT
groups, compared to their respective baselines (p < 0.0001) and control (p < 0.0001, Figure 4C). All
treatment groups at 7 days had elevated blood ketone levels, compared to their baselines (p < 0.0001)
and the control (p < 0.0001). At 7 days, the blood ketone levels in KEMCT and KS groups were
significantly increased from their 1-h level (p = 0.0025; p = 0.0007; respectively). Blood glucose levels
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decreased significantly at 1 h in the KE group, compared to baseline (p = 0.0307) and control (p = 0.0002),
in the KSMCT group, compared to baseline (p < 0.0001) and control (p < 0.0001), in the KEKS group,
compared to baseline (p = 0.0003) and control (p = 0.0022), and in KEMCT group, compared to baseline
(p < 0.0001) and control (p < 0.0001, Figure 4D). After 7 days, blood glucose levels decreased in KEKS
group, compared to baseline (p < 0.0001) and control (p = 0.0034), increased in KS and KSMCT groups,
compared to 1 h (p = 0.0006; p = 0.0006), and decreased in KEMCT group, compared to baseline
(p < 0.0001), and control (p < 0.0001).
3.4. Changes in Motor Performance, Blood Glucose, and R-βHB Levels in G1D Mice with Chronic Exposure
In G1D mice after chronic treatment the latency to fall on the accelerating rotarod was significantly
increased in the KE group at week 3 (p < 0.01), and at week 6 (p < 0.05), compared to baseline
(Figure 5A). The KD and KS groups were observed to have a trend of nonsignificant increase in latency
to fall. The percent change in latency to fall was not significantly elevated in any groups, compared
to baseline or control (Figure 5B). KD and KS were observed to have a marginal and nonsignificant
increase percent change in latency to fall, while the KE had a larger, but still nonsignificant increase
in percent change in latency to fall. Blood R-βHB levels increased significantly in the KD group at
week 6, compared to baseline (p = 0.0157) and control (p = 0.0211, Figure 5C). The KS group had a
significant increase in blood R-βHB levels at week 6, compared to baseline (p = 0.045). Blood glucose
level decreased significantly in the KE group at week 6, compared to baseline (p = 0.0113), and in the
KD group at week 3, compared to baseline (p = 0.0393, Figure 5D).
Figure 5. Changes in motor performance and blood parameters in postexercise state for G1D mice
with chronic treatment, at baseline, at week 3, at weeks 6, and at week 10. (A) The latency to fall
(sec) on accelerating rotarod presented, compared to baseline. (B) The percent change in latency to
fall at each timepoint, compared to baseline. (C) Blood R-βHB levels are presented in postexercise
state at each timepoint. (D) Blood glucose levels are presented in postexercise state at each timepoint.
Abbreviations: B: Baseline; 3: Week 3; 6: Week 6; 10: Week 10; R-βHB, R-beta-hydroxybutyrate; G1D,
Glucose Transporter Type-1 Deficiency Syndrome mice; KD, ketogenic diet; KE, ketone ester; KS,
ketone-salt. *: p < 0.05, **: p < 0.01 level of significance.
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4. Discussion
In this study, we demonstrated the effect of KD and various exogenous ketone formulations
on motor performance in different rodent models with (WR rats and G1D mice) and without (SPD
rats) pathology after acute, subchronic, or chronic exposures. We reported improvement of motor
performance by ketone supplementation, which may depend on the strain of rodents, specific ketone
formulation, age, species, and exposure frequency. Moreover, we reported significant changes in blood
R-βHB and glucose levels after exercise. Previous experiments have demonstrated that ketogenic
diets significantly improved motor function in studies on AD, ALS, HD, PD, stroke, and MS disease
models [1–7,9,33]. Our experiments expand upon these previous studies by reporting about the
positive effect of exogenous ketone supplementation on motor performance in nonpathological and
additional pathological rodent models.
Ketone-based therapies were found to be effective in the treatment of a variety of diseases,
including neurological pathologies with motor dysfunction [2,8,25,43–54]. For example, in patients
with PD nutritional ketosis decreased neuron degeneration, reduced mitochondrial damage, improved
motor outcomes, and reduced proinflammatory cytokine levels [1,4,43–46]. Application of nutritional
ketosis attenuated motor dysfunction in mouse models of HD [47]. Ketone-based therapies were
reported effective in improving motor function in a model animal of ALS (SOD1-G93A transgenic
mice) through neuroprotective outcomes [2,35]. Moreover, in a study on G1D mice (characterized
by impaired glucose transportation and motor dysfunction), adherence of a KD improved motor
symptoms [41]. Our results demonstrated that exogenous ketone supplement can improve motor
dysfunction in G1D mice. While several therapeutic mechanisms for the KD have been explored, its role
in disease pathologies is relatively unknown and further research into the physiological and mechanistic
effect of ketone-based therapeutics is warranted [1]. It has been demonstrated that exogenous ketone
supplementation is a viable and safe method in various animal models of, for example, central nervous
system (CNS) diseases and in human CNS disorders when alternative ketone-based therapies are
needed that address the limitations with KD [1,14,17,19,31].
We report that the motor performance may improve with KD in disease context, but also
with exogenous ketone supplementation even in rodents eating a standard, carbohydrate-rich diet,
suggesting that ketone supplementation may be a viable alternative for those unwilling or unable
to adhere to a KD. Our data supports that exogenous ketone supplementation was more efficient in
improving motor performance than KD, as KD reduced motor performance in SPD rats after 24 h and
7 days (Figure 3A), although the effect was not significant when assessing % change (Figure 3B). On
the other hand, exogenous ketones showed more consistent improvement throughout different model
systems, illustrating a potentially more translatable therapeutic tool across healthy and disease context.
While our previous study showed that ketone supplementation lowered blood glucose levels [27]
in the nonexercised state, we found that in the postexercise state it was elevated at 1 h (Figure 2D),
possibly due to exercise augmenting glycogenolysis induced glucose elevation which may offset the
blood glucose lowering effect. However, in some cases in SPD and WR rats after 1 h and 7 days the
blood glucose levels were lower than control and baseline (Figures 2D, 3D and 4D) and in G1D mice
after 6 weeks it was lower than baseline (Figure 5D), suggesting that exogenous ketones may be able to
offset the glycogenolysis induced glucose elevation in postexercised state. It has been suggested that
alleviating effects of ketone-based therapies, such as KD on motor performance, likely results from
carbohydrate restriction (and low glucose levels), but not from increased blood βHB levels [48,49].
However, based on our results it is unclear whether KD- and ketone supplements- evoked alterations
in blood glucose level had a major influencing factor on motor performance. For example, both KD
and KE decreased blood glucose level at 24 h (Figure 3D), but only KE improved motor performance of
SPD rats.
Many different beneficial effects on diseases have been linked to ketone therapies and this is likely
due to the resulting decreased production of reactive oxygen species (ROS), improved mitochondrial
function, reduction in inflammation, neuroprotective effects, and increased expression of brain-derived
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neurotrophic factor (BDNF) [1,50–52]. Additionally, ketones have been found to produce changes in
post-translational modification of proteins and histone acetylation [53–55]. It has also been reported
that ketones may enhance learning in multiple rodent models and may explain the motor performance
increase observed in the nonpathological mouse models [56]. Furthermore, ketones may enhance
muscle performance or CNS control of muscle contractions/coordination, which could be a contributing
factor to the rodent’s ability to remain on the rotarod longer, not only in rodents with motor dysfunction,
but also rodents without motor impairment, such as SPD rats and WR rats.
Ketogenic diet and exogenous ketone supplementation may also act on cellular homeostasis
through various signaling pathways, such as mTOR, AMPK, and neurotransmitter systems. Ketosis has
been shown to affect cell proliferation, energetic metabolism, protein biosynthesis, and attenuate muscle
wasting [57–62]. KD acts upon specific tyrosine kinase receptors for insulin and insulin growth factor
(IGF) and upregulates the phosphatidylinositol-3 kinase (PI3K)-Akt-mammalian target of rapamycin
complex 1 (mTORC1). This is counteracted by a lower intracellular ATP/AMP ratio and the subsequent
upregulation of liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) signaling, which then
inhibits the mTORC1 pathway [48,53]. Research also suggests that ketone-based therapies likely have
additional therapeutic applications by reduced levels of proinflammatory cytokines, ROS, attenuation
of skeletal muscle catabolism, and various neuroprotective effects [15,50,51,62,63]. Moreover, it was
also suggested that KD- and exogenous ketone supplements-evoked alleviating effects on different
CNS diseases may be modulated by different neurotransmitter/neuromodulator systems, such as
GABAergic (e.g., by increased GABA level) and purinergic/adenosinergic (e.g., via increased adenosine
concentration) systems [19,31,64–67]. Therefore, studies about the application of ketone metabolic
therapies for various neurological conditions suggest that multifactorial mechanisms play a role in
improving motor function (e.g., mitochondrial function, neurotransmitter systems, anti-inflammatory
processes, and skeletal muscle physiology), but further research is required to fully elucidate pathways
and potential therapeutic applications.
While no motor function impairments (motor dysfunction) have been demonstrated in SPD
rats and WR rats [39,40], the subchronically administered ketone supplement KE, KS (in SPD rats),
KEKS and KEMCT (in WR rats) improved motor performance. These results suggest that exogenous
ketone supplements may improve motor performance not only in animal models with neurometabolic
pathology, such as motor dysfunction (e.g., G1D mice), but also in animals without pathology (e.g.,
SPD rats) or with pathology, but without motor dysfunction (e.g., WR rats), likely by enhanced learning
and muscle performance [5,44,56,68]. Moreover, exogenous ketone supplements-evoked effects on
motor performance may be rat strain dependent. Indeed, it was demonstrated that influences of KD
and ketone supplements may be modulated by strain-, age-, and species-dependent changes. For
example, it has been demonstrated that expression of monocarboxylate class type 1 transporter on the
BBB may be not only age-, but also species-dependent [69–71]. In addition, age- and species-dependent
changes in activity of βHB metabolizing enzymes were also demonstrated [39,40,72–75]. It has been
revealed that level, uptake, metabolism, as well as utilization, and consequently, effects of ketone
bodies may be regionally different in the brain areas implicated not only in physiological, but also in
pathophysiological processes in different strains [72,76–78]. All of these factors may result in brain area-,
age-, strain-, and species-dependent influences/effectivity of KD and ketone supplements on motor
performance in different animal models. Consequently, efficacy of treatments may also be dependent
on the various type of ketone supplements (e.g., KSMCT decreased whereas KEKS and KEMCT
increased the motor performance in WR rats), on the exposure frequency/time after administration
(e.g., acute treatment by KE was not effective on motor performance, but subchronic administration of
KE improved the motor performance in SPD rats) and on the interference of different factors.
Limitations and Inconsistencies

One limitation of these results is that we used racemic (R/S) βHB exclusively, because this was the
most economical and commercially available product on the market for both experimental purposes
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and for consumer consumption at the time of the study. Racemic βHB has been used clinically
in patients and consumers for years [79], and no serious adverse events from βHB supplement
ingestion have been reported (CAERS open-FDA database), even with >1 million estimated servings
consumed annually. Thus, our approach was to evaluate molecules, which may be economically
feasible to translate to humans and would be accessible to end-users in the near future. However, one
consideration with racemic βHB supplements is the potential differences in metabolism of S- βHB
and R- βHB, although existing data on this is still limited. Based on previous studies we know that S-
βHB and R- βHB from racemic mixtures are naturally metabolized in rats and humans [80], but S-βHB
remains elevated in the blood longer after consumption of racemic mixtures. It appears that ketolytic
metabolism favors a more rapid catabolism of R- βHB, the predominant form of βHB that is produced
endogenously. It is possible that the rate limiting enzymes for S-βHB conversion to acetyl CoA (e.g.,
S-3-hydroxybutyryl-CoA dehydrogenase) may be in lower quantities since endogenous levels are much
lower. Although previous studies suggest that the enzymatic kinetics of S- βHB metabolism is slower,
this does not appear to be the case in the rat liver, at least with S-1,3-butanediol. Of potential relevance
to our results is the observation that S-1,3-butanediol, a specific S- βHB precursor, has a greater blood
glucose lowering effect [81], which could conceivably influence motor function performance. These
previous metabolic observations need to be considered when interpreting our data and designing
future experiments that test enantiomerically pure forms of both βHB isomers in physiological testing.
Considering these limitations, we can speculate that R-βHB levels may be an important factor in
KD/exogenous ketone supplement-evoked positive effects on motor performance, but other factors
likely have a modulatory role. Since we could not unambiguously correlate elevated R-βHB levels
with augmented motor performance, future dose-response studies can help to identify the optimal
range for this specific application. It is also possible that other forms of ketone molecules contributed
to the performance enhancing effect that were not directly measured in the present study, but previous
studies show their elevation (e.g., KE will elevate acetoacetate levels as well, which was not directly
measured here, but may have contributed to the performance enhancing effect, see Figure 5B,C).
For example, acute KSMCT and KEKS treatment significantly increased the blood R-βHB level at 1 h,
but did not augment motor performance (latency to fall), whereas KEMCT treatment enhanced R-βHB
concentration to a lesser degree (compared to KSMCT and KEKS), but significantly increased latency
to fall, compared to baseline in one year old SPD rats (Figure 2). Acetoacetate, from KE, may have
contributed to the performance enhancing effect here as well, which may have increased efficacy for this
application when working synergistically with MCT rather than with KS. In regards to KD treatment,
we observed increased blood R-βHB at 24 h and 7 days, but this treatment significantly decreased
the latency to fall from rotarod, compared to baseline in 4-months-old SPD rats (Figure 3). It is likely
that adaptation (which takes weeks to months) to the KD confers additional beneficial effects, so this
outcome was not unexpected after only 24 h and even 7 days, as we would expect to see ketoadaptation
and its beneficial effects over longer timeframe. KE treatment significantly increased blood R-βHB
level only at 24 h, but improved motor function at both 24 h and 7 days, compared to baseline (24 h and
7 days), and control (at 7 days). KS treatment did not increase blood R-βHB levels at 24 h, however this
treatment significantly improved motor function, compared to both control and baseline (Figure 3). It
is conceivable that low R-βHB at 24 h is indicative of tissue utilization, leading augmentation of muscle
tissue performance. Moreover, KSMCT treatment increased blood R-βHB concentration to high levels
at 7 days more than any other treatment, but it did not show a motor performance improving effect
(Figure 3), suggesting that higher R-βHB levels may not be as beneficial, or that MCT may be negating
the performance benefits (perhaps by reducing R-βHB metabolism). Subchronic treatment of WR rats
by KE and KSMCT increased blood R-βHB levels at 1 h and at 7 days, but did not improve motor
performance (Figure 4), again suggesting that higher R-βHB levels might not be optimal in this context.
Chronic KE treatment improved motor performance at week 3 and week 6 in G1D mice, but blood
R-βHB levels were not changed by KE treatment at these time points (Figure 5). Therefore, it is possible
that other ketone bodies, such as acetoacetate may contribute to the performance enhancing effect that
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was associated with chronic KE treatment. Based on these results, we can speculate that a particular
range of R-βHB level may be needed to improve motor function, and this may be age-, strain-, and
species dependent. It is possible that higher levels of acute (or subchronic) ketosis may induce a mild
metabolic acidosis (lower pH), and this could conceivably blunt performance effects. Moreover, other
contributing factors should also be considered, e.g., different ketone-based therapies evoke direct and
indirect influences on neurotransmission, cell energetics, muscle and nerve regeneration, as well as
gastric hormone levels (e.g., ghrelin, which is in connection with muscular trophism) [48,82,83], other
modulatory effects, such as mobilization of polyunsaturated fatty acids (PUFAs),which can directly
modulate ion channel functioning [84,85], and elevated neurosteroid synthesis resulting in modulation
of GABAA receptors [86,87]. These and other, yet unidentified factors may influence the modulatory
role of KD/ketone supplements on physiological and pathophysiological processes, as well as CNS
diseases. Thus, our results suggest that KD/ketone supplements may be effective to improve motor
function in different physiological and pathophysiological conditions by affecting multiple organs.
Given these inconsistencies we need to point out the following: (i) we could not make definitive
conclusion about direct correlation between motor performance and R-βHB levels; (ii) our experimental
design did not mechanistically elucidate the contributing role of acetoacetate (and perhaps acetone) in
augmenting motor performance; (iii) these data do not clarify if ketone supplementation augments
performance via enhancing muscular cellular energetics or through some yet to be identified
ketone-induced signaling/neuropharmacological change, potentially influencing CNS-control of motor
performance. However, we can conclude that (i) positive effects of exogenous ketone supplementation
were identified on motor performance in nonpathological and pathological states; (ii) exogenous
ketones may be able to offset the glycogenolysis induced glucose elevation in postexercised state;
(iii) exogenous ketones may have a specific therapeutic range for various applications; (iv) different
forms and formulations can have different effects independently from the level of blood βHB elevation.
In order to address these remaining questions, future experiments will need to focus on:
(1) More direct comparisons of the physiological effects between racemic and enantiomerically
pure βHB substances; (2) Comprehensively evaluate the dose-dependent effects of both racemic and
enantiomerically purified forms of βHB; (3) Investigate acetoacetate-specific effects; (4) Monitoring
correlations between blood glucose lowering effect and motor performance; (5) Conduct additional tests
focusing on potential differences in efficacy between various motor functions, e.g., endurance, fine motor
control, strength, balance; (6) Mechanistically isolate R-βHB signaling effects from ketone-induced
changes in cellular energetics and metabolic control, especially focusing on signaling effects that can
influence motor performance; (7) Detailed investigation of agent-specific mechanism on not only
the brain, but also on other organs (e.g., heart, skeletal muscle, lungs, and liver); (8) Develop and
identify optimal ketone formulations (e.g., types, doses, exposure frequency vs. age) to alleviate motor
dysfunction in patients with different physiological and pathological conditions.
In light of these inconsistencies in the data we would like to caution end-users to avoid making
generalizations about the use of ketone supplements in different contexts and we call to direct
more attention to the differences between various forms (racemic and enantiomerically pure) and
formulations (combined agents) and their potential positive, negative, or synergistic effect.
5. Conclusions
Our study demonstrates the effects of the KD and ketone supplementation on motor performance
in SPD, and WR rats, as well as in G1D mice after acute, subchronic, or chronic administration. We
observed that motor performance improved significantly in various pathological and nonpathological
rodent models for different exposure schedules and that the effectiveness of the supplements differed
according to rodent strain, the schedule of treatment, the specific supplement they were exposed
to, and age of the rodents. We found that exogenous ketone supplementation was more efficient in
improving motor performance than KD. While KD treatment led to mixed results—demonstrating
more efficacy in disease context (G1D)—exogenous ketones showed consistent effect across disease
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and nondisease states. Furthermore, in certain scenarios, exogenous ketone supplements offset
the glycogenolysis-induced glucose elevation in the postexercise state, which might be beneficial
when there is a need to keep blood glucose levels low even after exercising. These results also
strengthen previous research on the potential of ketone therapies and their ability to improve motor
performance in a variety of pathological and nonpathological rodent models. The differences in
the efficacy of a given formulation are potentially due to differences in neuronal ketone metabolism
between the different rodent strains and species, hepatic metabolism (ketogenesis), or variabilities in
skeletal muscle metabolism. However, further studies are needed to explore the safe and effective
therapeutic applications of ketone-based therapies and elucidate their exact mechanisms of action on
motor performance.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/8/2459/s1,

Table S1: NIH-31 Open Formula Mouse/Rat Sterilizable Diet, Table S2: Teklad Custom Research Diet Data Sheet.
Author Contributions: Conceptualization, C.A.; Data curation, C.A.; Formal analysis, C.A.; Funding acquisition,
C.A., D.P.D. and Z.K.; Investigation, C.A., C.M., C.G., A.P.K., S.R.B. and Z.K.; Methodology, C.A. and Z.K.; Project
administration, C.A. and Z.K.; Resources, D.P.D. and Z.K.; Supervision, C.A.; Writing–original draft, C.A., S.B.
and Z.K.; Writing–review and editing, C.A., A.P.K., S.R.B., D.M.D., M.K., D.P.D. and Z.K. All authors have read
and agreed to the published version of the manuscript.
Funding: Funding was provided by Quest Nutrition (to Csilla Ari), ONR Grant N000141310062 (to
Dominic P. D’Agostino), the Glucose Transporter Type-2 Deficiency Syndrome Foundation (Glut1DS)
(to Dominic P. D’Agostino), and the National Development Agency of Hungary (under Grant No.
TIOP-1.3.1.-07/2-2F-2009-2008), and OTKA K124558 Research Grant (to Zsolt Kovács).
Acknowledgments: Quest Nutrition provided partial funding to Csilla Ari.
Conflicts of Interest: International Patent # PCT/US2014/031237, University of South Florida for DPD:
Compositions and Methods for Producing Elevated and Sustained Ketosis. Patent pending: USF Ref. No:
16A019 for CA and DPD: “Exogenous Ketone Supplementation Improved Motor Function in Sprague-Dawley
Rats.” DPD and CA are co-owners of the company Ketone Technologies LLC, a company specialized on scientific
consulting. DMD is a paid consultant and member of the science advisory board for Axcess Global Sciences and
Anutra. These interests have been reviewed and managed by the university in accordance with its Institutional
and Individual Conflict of Interest policies. All authors declare that there are no additional conflicts of interest.
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nutrients
Review
Effects of Beta-Alanine Supplementation on Physical
Performance in Aerobic–Anaerobic Transition Zones:
A Systematic Review and Meta-Analysis
Álvaro Huerta Ojeda 1, *, Camila Tapia Cerda 2 , María Fernanda Poblete Salvatierra 2 ,
Guillermo Barahona-Fuentes 1 and Carlos Jorquera Aguilera 3
1 Grupo de Investigación en Salud, Actividad Física y Deporte ISAFYD, Escuela de Educación Física,
Universidad de Las Américas, sede Viña del Mar 2531098, Chile; [email protected]
2 Facultad de Ciencias, Escuela de Nutrición y Dietética, Magíster en Nutrición para la Actividad Física
y Deporte, Universidad Mayor, Santiago 8580745, Chile; [email protected] (C.T.C.);
[email protected] (M.F.P.S.)
3 Facultad de Ciencias, Escuela de Nutrición y Dietética, Universidad Mayor, Santiago 8580745, Chile;
[email protected]
* Correspondence: [email protected]; Tel.: +56-9-77980432
Abstract: Beta-alanine supplementation (BA) has a positive impact on physical performance. However,
evidence showing a benefit of this amino acid in aerobic–anaerobic transition zones is scarce and the
results controversial. The aim of this systematic review and meta-analysis is to analyze the effects of
BA supplementation on physical performance in aerobic–anaerobic transition zones. At the same
time, the effect of different dosages and durations of BA supplementation were identified. The search
was designed in accordance with the PRISMA® guidelines for systematic reviews and meta-analyses
and performed in Web of Science (WOS), Scopus, SPORTDiscus, PubMed, and MEDLINE between
2010 and 2020. The methodological quality and risk of bias were evaluated with the Cochrane
Collaboration tool. The main variables were the Time Trial Test (TTT) and Time to Exhaustion
(TTE) tests, the latter separated into the Limited Time Test (LTT) and Limited Distance Test (LDT).
The analysis was carried out with a pooled standardized mean difference (SMD) through Hedges’ g
test (95% CI). Nineteen studies were included in the systematic review and meta-analysis, revealing a
small effect for time in the TTT (SMD, −0.36; 95% CI, −0.87–0.16; I2 = 59%; p = 0.010), a small effect
for LTT (SMD, 0.25; 95% CI, −0.01–0.51; I2 = 0%; p = 0.53), and a large effect for LDT (SMD, 4.27;
95% CI, −0.25–8.79; I2 = 94%; p = 0.00001). BA supplementation showed small effects on physical
performance in aerobic–anaerobic transition zones. Evidence on acute supplementation is scarce
(one study); therefore, exploration of acute supplementation with different dosages and formats on
physical performance in aerobic–anaerobic transition zones is needed.
Keywords: beta-alanine; ergogenic aid; physical performance; aerobic–anaerobic transition zone
1. Introduction
A proper diet is one of the main factors in the improvement of physical performance. However,
sometimes it is not enough to meet the energetic demands of training sessions [1]. For this reason and
with the aim of maximizing physical performance, the use of nutritional supplements is widespread
in sport [2], even more in younger athletes [3]. Nutritional supplements, such as protein and
carbohydrates, are concentrated nutrient sources that substitute or complement the use of certain
foods, while ergogenic aids, such as caffeine, creatine, or beta-alanine (BA), are pharmacological agents
used with the aim of enhancing physical performance [4]. In this regard, one study showed that
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Nutrients 2020, 12, 2490
48% of athletes use nutritional supplements and ergogenic aids [3], claiming that certain components,
such as creatine, caffeine, sodium bicarbonate, and BA, contribute to an improvement in their physical
performance [5–7].
Specifically, BA is a non-essential amino acid synthesized in the liver and found in products of
animal origin [8]. Evidence shows that poultry, beef, and fish are products with a large BA content [9].
BA has been consistently shown to increase levels of carnosine (CA) in human skeletal muscle [9–12].
This last substance is synthesized by CA synthase when bonding BA with L-histidine [13]; CA is found
in the muscular tissue and acts as a buffer of hydrogen protons (H+ ) in high-intensity physical exercises
of short duration [11,14]. This is why athletes who follow a vegetarian diet will have lower muscular
CA concentrations than those who follow an omnivorous diet [15].
When performing high-intensity exercises, due to the predominant energetic system (anaerobic
metabolism of carbohydrates), a high release of H+ takes place, which leads to a decrease in pH [12].
This pH decrease can negatively affect the metabolic processes of phosphocreatine resynthesis,
inhibit contractile processes, and diminish the glycolytic rate—all these factors contribute to the onset of
muscular fatigue [14]. Some studies have concluded that an elevated muscular CA concentration could
buffer between 8–15% of H+ , opening the possibility of maximizing physical effort for a longer period
of time [1]. On the other hand, other studies have shown that CA and L-histidine supplementation do
not increase the bioavailability of intramuscular CA [5,14]. For this reason, and considering BA as
a precursor in CA formation, several studies have shown an increase between 40–80% of intramuscular
CA post BA supplementation [1,9–11,16]. In this regard, the acute effect of BA supplementation
has been tested in doses of 30 mg·kg−1 of body mass and prolonged supplementation with doses
ranging from 2.0 to 6.4 g/day for periods of time between 4 and 10 weeks [12,17]. At the same time,
BA can be found as the main ingredient in multi-ingredient pre-workout supplements, although
it is worth mentioning that these products have a lower dosage than that studied clinically [18].
Specifically, lower pH values have been measured after 4 min of high-intensity exercise [19] and the
drop of pH is one of the factors responsible for the increase in ventilatory responses [20]. In parallel,
the background shows that BA supplementation reported only one secondary effect, paresthesia [21,22];
this is a sensation of flushing associated with an irritant tingling in the ears, scalp, hands, and torso [23].
Related to pH stabilization, there are several studies that have used ergogenic aids to improve
physical performance in aerobic–anaerobic transition zones [8,24]. The aerobic–anaerobic transition
zone corresponds to an intensity range between aerobic threshold and anaerobic threshold [25] and
may serve as a basis for assessing endurance performance individually as well as for prescribing
intensities in endurance training [26]. In this regard, BA is among the ergogenic aids used to increase
performance in aerobic–anaerobic transition zones [8,9,27]. One study has evaluated the effect of
BA supplementation on physical performance, showing an improvement of 13.9% in ventilatory
threshold [20]. In addition, another study reported that BA supplementation for 28 days enhanced
sub-maximal endurance performance by delaying the onset of blood lactate accumulation (OBLA) [8].
However, other investigations have not found significant results in athletic performance [12], specifically
in rowers [28], and trained cyclists [29] with BA supplementation.
The existing evidence shows controversial results that make it impossible to categorize or
ensure that BA supplementation improves physical performance in aerobic–anaerobic transition zones
(performance mainly connected to ventilator parameters). Hence, the primary aim of this systematic
review and meta-analysis was to analyze the effects of BA supplementation on physical performance
in aerobic–anaerobic transition zones. Likewise, the effects of different doses and supplementation
times with BA were identified.
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Nutrients 2020, 12, 2490
2.1. Literature Search Strategies

In order to perform this review, a thorough electronic search was carried out in several databases
and search engines. Articles published in Web of Science (WOS), Scopus, SPORTDiscus, PubMed,
and MEDLINE were included. A search limit was established from January 2010 to February 2020.
The bibliographic search was performed in accordance with the PRISMA® statement guidelines
for systematic reviews and meta-analyses [30]. In each of the aforementioned databases, the search
included hits in the title, abstract, and key words search fields. The following key words were combined
with Boolean operators AND/OR: [(“b-alanine” OR “beta-alanine” OR “b-alanine supplementation” OR
“beta-alanine supplementation”) AND (“maximal aerobic speed” OR “maximal oxygen uptake” OR
“maximal aerobic consumption” OR “endurance”)]. One of the authors performed the search, and two
reviewed the studies. Together, they decided whether the studies were appropriate for inclusion.
2.2. Inclusion and Exclusion Criteria

The importance of each study was assessed according to the following inclusion criteria: (1) BA
supplementation, either acute or chronic supplementation, (2) experimental design studies, (3) healthy
adults, (4) studies that included physical performance evaluation in the aerobic–anaerobic transition
zone (60–100% VO2 max), (5) studies that included Time Trial Tests (TTT), or Time to Exhaustion
(TTE) tests for physical performance evaluation, (6) studies that stated a baseline and control group,
(7) studies showing negative and positive changes in TTT or TTE tests, and (8) studies published in
English and Spanish. The studies that failed to fulfill the inclusion criteria were not considered in the
systematic review nor the meta-analysis. Possible discrepancies were resolved through discussion
until a consensus was reached.
2.3. Chronic and Acute Supplementation

Regarding the classification of the supplementation protocols assessed in this systematic review,
acute supplementation was considered to be the one in which a unique dose of BA was used between
0 min and 24 h prior to physical exercise, while chronic supplementation was considered those protocols
that used repeated dosages of BA for more than one day and up to 10 weeks [31].

The articles were examined regarding the effect of BA supplementation on physical performance
in aerobic–anaerobic transition zones (60–100% VO2 max) [26,32]. The primary outcome used for the
systematic review and meta-analysis were (a) TTT and (b) TTE tests (Limited Time Test (LTT) and
Limited Distance Test (LDT)). In order to establish the upper limit in the aerobic–anaerobic transition
zone (100% VO2 max), the minimum time used on the TTT and TTE test (LTT) was 300 s (the literature
sets this as the minimum amount of time needed to determine VO2 max) [33,34], while the minimum
lower limit in the aerobic–anaerobic transition zone was 60% of VO2 max [32]. These limits were set in
order to include studies showing results of 5–63 min [21,35]. The systematic review and meta-analysis
also included secondary outcomes stated in the studies. These secondary variables were (a) capillary
lactate (mmol·L−1 ), (b) absolute VO2 max (LO2 ·min−1 ), (c) HR (bpm), and (d) ratings of perceived
exertion (RPE) according to the Borg scale [36]. It is important to mention that studies were excluded
from the systematic review and meta-analysis if they only showed secondary results in the in extenso
reading. Median values, standard deviations (SD), and sample sizes were included for the statistical
analysis of the meta-analysis, for both the primary and secondary outcomes. If the selected studies
did not include numerical data, it was requested of the authors, or if the data were plotted as figures,
the values were estimated based on the pixel count. Additionally, the studies that declared paresthesia
symptoms in their subjects were also included.
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Nutrients 2020, 12, 2490
2.5. Publication Bias

Publication bias was assessed using Egger’s statistical test. This test determined the presence of
bias at p ≤ 0.05 [37]. Funnel plots were created to interpret the general effect, followed by an Egger’s
statistic to confirm or refute publication bias. Egger’s analysis suggested that the primary variables
did not show publication bias: (a) TTT: z = 1.35, p = 0.18; (b) LTT: z = 1.90, p = 0.06; (c) LDT: z = 1.85,
p = 0.06 (Figure 1).
ȱ ȱ ȱ
(a)ȱ (b)ȱ (c)ȱ
Figure 1. Standard error for Times Trial Test (a), Limited Time Test (b), and Limited Distance Test (c).
SE: standard error; SMD: standardized median difference.
2.6. Quality Assessment of the Experiments

The methodological quality and risk of bias for each selected study were assessed through
a Cochrane Collaboration guideline [38]. The list was divided into six different domains: selection bias
(random sequence generation, allocation concealment), performance bias (blinding of participants and
personnel), detection bias (blinding of outcome assessment), attrition bias (incomplete outcome data),
reporting bias (selective reporting), and other types of bias (declaration of conflict of interest). For each
item, the answer to a question was considered; when the question was answered with a “Yes”, the bias
was low; when it was “No”, the bias was high; when it was “Unclear”, the possible bias was connected
to a lack of information or uncertainty. The full details of each study and domains are presented in
Figures 2 and 3.
ȱ
Figure 2. Risk of bias graph: review authors’ judgements about each risk of bias item presented as
percentages across all included studies.
80
Nutrients 2020, 12, 2490
ȱ
Figure 3. Risk of bias summary: review authors’ judgements about each risk of bias item for each
included study.

In order to evaluate the quality of the experiments and interpret the risk of bias values,
Review Manager version 5.4 was used (Copenhagen: The Nordic Cochrane Centre, The Cochrane
Collaboration, 2014). The same software was used to perform a descriptive and statistical analysis
of the meta-analysis. To compare the supplementation of BA versus the placebo (PL), the number
of participants, standardized mean difference (SMD), and standard error of SMD were analyzed for
each study. Hedges’ g test was used to calculate the SMD of each study [39]. The overall effect and its
95% confidence interval (CI) were calculated by weighting the SMD by the inverse of the variance.
Additionally, the SMD of both the BA supplemented and PL groups were subtracted to obtain the
net effect size (ES), which was used together with the pooled SD of change to calculate the variance
(ES = [mean BA − mean PL]/SD); to interpret the magnitude of the ES, Cohen’s criteria were followed:
<0.2, trivial; 0.2–0.5, small; 0.5–0.8, moderate; and >0.8, large [40].
The I2 statistic was calculated as an indicator of the percentage of observed total variation within
studies due to real heterogeneity rather than chance. I2 values are included from 0 to 100%, representing
a small amount of inconsistency between 25% and 50%, a medium amount of heterogeneity between
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Nutrients 2020, 12, 2490
50% and 75%, and a large amount of heterogeneity when the I2 value was higher than 75%. In this sense,
low, moderate, and high adjectives would be accepted referring to I2 values of 25%, 50%, and 75%,
respectively, although a restrictive categorization would not be adequate in all circumstances [41].
3. Results
3.1. Main Search

The literature search through electronic databases identified 323 articles of which 177 were
duplicates. The remaining 146 articles were filtered by title and abstract, and 57 studies remained to
be read and analyzed. After a review of those 57 studies, 40 were eliminated because they did not
meet the inclusion criteria. In the search for articles oriented by bibliographic references, two extra
studies were added. As a result, 19 articles were included for the systematic review and meta-analysis.
The search strategy and study selection are shown in Figure 4. Out of 19 studies, eight considered time
in a TTT to assess the effect of BA supplementation on physical performance [29,42–48], nine used time
(LTT) on a TTE test to assess the same effect [5,21,22,35,49–53], one considered the distance (LDT) on
a TTE test [28], while one considered both the time (LTT) and the distance (LDT) on a TTE test [17]
(Table 1).
3.2. Effect of BA on Time Trial Tests

Eight studies were considered for this analysis [29,42–48]. However, two of them included two
TTTs in the research design [29,44]. For the meta-analysis, the study by Bellinger et al. [29] was
considered as two independent designs (TTT of 4 and 10 km on a cycle ergometer, respectively). In the
same way, the study by Bellinger et al. [44] was considered as two independent designs (TTT of 4 and
10 km on a cycle ergometer, respectively). Thus, 10 studies were included in the meta-analysis that
calculated the effect of BA supplementation on time in TTT. Figure 5 shows that BA supplementation
generates a small and non-significant effect on physical performance in TTT (SMD, −0.36; 95% CI
−0.87–0.16; p = 0.18). The meta-analysis showed moderate heterogeneity among the studies reviewed
(I2 = 59%; p = 0.01). Out of the 10 studies analyzed, seven of them declared a beneficial effect of
supplementation with BA on physical performance in TTT [29,42,43,46–48]. Out of these studies, the
research of Santana et al. [48] presented a large ES (−6.70). On the other hand, three of the 10 studies
showed a neutral or prejudicial effect after BA supplementation [44,45].
3.3. Effect of BA on the Limited Time Test

Ten studies were considered for this analysis [5,17,21,22,35,49–53]. However, one of them
included two experimental groups for the LTT in their research design [21]. For the meta-analysis,
two experimental groups presented by Smith-Ryan et al. [21] were considered as two independent
studies (LTT at 90% of VO2 max on a treadmill for women and LTT at 90% of VO2 max on a treadmill
for men). This way, 11 studies were included in the meta-analysis that calculated the effect of BA
supplementation on the TTE test. Figure 6 shows that BA supplementation generated a small and
non-significant effect for time on the TTE test (SMD, 0.25; 95% CI −0.01–0.51; p = 0.06). A meta-analysis
showed low heterogeneity among the reviewed studies (I2 = 0%; p = 0.53). Out of the 11 studied and
analyzed, eight showed a positive effect of BA on time in the LTT [5,17,21,22,49–52]. Out of these
studies, Furst et al. [49] showed a large ES (1.64). On the other hand, three of the 11 studies showed an
unbeneficial effect after BA supplementation [21,35,53].
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Recordsȱidentifiedȱthroughȱ
databaseȱsearchingȱ
Identificationȱ
(nȱ=ȱ323)ȱ
Duplicatedȱrecordsȱ(nȱ=ȱ177)ȱ
Recordsȱexcludedȱbyȱtitlesȱandȱ
Recordsȱafterȱduplicatesȱ
abstractsȱ
Screeningȱ
removedȱ(nȱ=ȱ146)ȱ ȱ
(nȱ=ȱ89)ȱ
Recordsȱexcludedȱwithȱreasonsȱ
(nȱ=ȱ40):ȱ
x Noȱevaluationȱofȱprimaryȱ
FullȬtextȱarticlesȱassessedȱ outcomes:12ȱ
Eligibilityȱ
forȱeligibilityȱ x Typeȱofȱtests:15ȱ
(nȱ=ȱ57)ȱ x MultiȬingredientȱ
supplementation:ȱ11ȱ
x Noȱsupplementationȱwithȱ
betaȱalanina:ȱ2ȱ
Additionalȱrecordsȱidentifiedȱ
Includedȱandȱanalyzedȱ
throughȱotherȱsourcesȱ
(nȱ=ȱ2)ȱ
Studiesȱincludedȱandȱ
analyzedȱinȱsystematicȱ
reviewȱandȱmetaȬanalysisȱ
(nȱ=ȱ19)ȱ
ȱ
Figure 4. Studies included in the systematic review and meta-analysis.
83
Table 1. Characteristics of the studies that connect BA supplementation with physical performance in aerobic–anaerobic transition zone.
Supplementation Performance
Author Objective Subjects Variables Test Results
Protocol in PO
Chronic effect of BA supplementation in aerobic–anaerobic transition zone
I:
To investigate if
C/A: Rowers EG: BA + Oral suppl of 7 w
performance is related Time (s) post test:
Nutrients 2020, 12, 2490
M = 16; F = 1 training TTT: 2000 m Total dose 7 w: 245 g

to the muscle CA EG = 386.5 vs. CG = 391.5; p > 0.05
Baguet et al. [42] (EG = 8; CG = 9) CG: PL + rowing EG: BA 5 g/day (5 doses ↔
content and if BA suppl The authors declared paresthesia:
A: 22.9 ± 4.2 training ergometer of 1 g, c/2 h)
improves performance no
years D: CG: maltodextrin
in highly trained rowers.
PO: TTT
Distance (m) post test:
GE1 = 7579 vs. CG = 7228; p > 0.05
GE2 = 7575 vs. CG = 7228; p > 0.05
VO2 max (LO2 ·min−1 ) post test:
I: GE1 = 3.63 vs. CG = 3.33; p > 0.05
EG: BA + Oral suppl of 28 d GE2 = 3.50 vs. CG = 3.33; p > 0.05
RA: Rowers
To investigate the effect training Total dose 28 d: 67.2 g [La] (mmol·L−1 ) post test:
M = 27
of two BA dosing CG: PL + LDT: 30 min EG1 : BA 2.4 g/day (1 GE1 = 10.0 vs. CG = 9.1; p > 0.05
Beasley et al. (EG1 = 9; EG2 =
strategies on 30 min training rowing dose of 2.4 g, e/24 h) GE2 = 8.8 vs. CG = 9.1; p > 0.05 ↔
[28] 9; CG = 9)
rowing and subsequent D: ergometer EG2 : BA 4.8 g/day (1 RPE (1–10) post test:
A: 24.0 ± 5.0
84
sprint performance. PO: LDT dose of 4.8 g, e/48 h) GE1 = 9.7 vs. CG = 9.6; p > 0.05
years
SO: VO2 max, CG: corn flour GE2 = 9.3 vs. CG = 9.6; p > 0.05
[La], RPE, HR HR (bpm) post test:
GE1 = 190 vs. CG = 185; p > 0.05
GE2 = 182 vs. CG = 185; p > 0.05
The authors declared paresthesia:
no
To investigate the effects
of BA suppl on the Time (s) post test:
I:
resultant blood acidosis, EG = 355.6 vs. CG = 360.4; p < 0.05
EG: BA + Oral suppl of 28 d
lactate accumulation, VO2 max (LO2 ·min−1 ) post test:
C/A: Cyclists training Total dose of 28 d: 179.2
and energy provision EG = 4.45 vs. CG = 4.44; p > 0.05
M = 17 CG: PL + g
Bellinger et al. during TTT 4000 m cycle [La] (mmol·L−1 ) post test:
(EG = 9; CG = 8) training EG: BA 6.4 g/day (4 dose ↑
[43] supramaximal-intensity ergometer EG = 15.1 vs. CG = 15.2; p > 0.05
A: 24.5 ± 6.2 D: of 1.6 g, in every meal)
cycling, as well as the RPE (6–20) post test:
years PO: TTT CG: dextrose
aerobic and anaerobic EG = 18.8 vs. CG = 18.8; p > 0.05
SO: VO2 max, monohydrate
contribution to power The authors declared paresthesia:
[La], RPE
output during a 4000 m no
cycling time trial.
Table 1. Cont.
Protocol in PO
4 km
Time (s) post test:
To assess the efficacy of
Nutrients 2020, 12, 2490
I: EG = 356.6 vs. CG = 357.8; p > 0.05

BA suppl on cycling
EG: BA + Oral suppl of 28 d [La] (mmol·L−1 ) post test:
time trial of different C/A: Cyclists
training Total dose 28 d: 179.2 g EG = 15.3 vs. CG = 15.4; p > 0.05
length in the same M = 14
Bellinger et al. CG: PL + TTT 4 km and 10 km EG: BA 6.4 g/day (4 dose 10 km 4 km ↔
group of trained cyclists (EG = 7; CG = 7
[29] training cycle ergometer of 1.6 g, with every meal) Time (s) post test: 10 km ↔
and to contrast the A: 24.8 ± 6.7
D: CG: dextrose EG = 938.1 vs. CG = 929.9: p > 0.05
effects of BA supply on a years
PO: TTT monohydrate [La] (mmol·L−1 ) post test:
supramaximal time to
SO: [La] EG = 11.0 vs. CG = 12.9; p > 0.05
fatigue test.
yes
To investigate the effects
of BA suppl only, and in Oral suppl of 9 w
4 km
combination with I: Total dose 9 w: 221.2 g
Time (s) post test:
sprint-interval training, C/A: Cyclists EG: BA + EG: BA 6.4 g/day for 4 w
EG = 339.7 vs. CG = 350.1; p > 0.05
on training intensity, M = 14 training (4 doses of 1.6 g,
Bellinger et al. TTT 4 km and 10 km 10 km 4 km ↔
and energy provision (EG = 7; CG = 7) CG: PL + w/every meal) + BA 1.2
85
[44] cycle ergometer Time (s) post test: 10 km ↔
and performance during A: 25.4 ± 7.2 training g/day for 5 w (3 doses of
EG = 918.5 vs. CG = 916.6; p > 0.05
exhaustive years D: 400 mg, every 3 to 4 h)
supramaximal-intensity PO: TTT CG: dextrose
no
cycling and a 4 and 10 monohydrate
km time trial.
Time (s) post test:
I:
To investigate whether EG = 3696 vs. CG = 3780; p > 0.05
C/A: Cyclists EG: BA +
BA suppl can increase Oral suppl of 6 w [La] (mmol·L−1 ) post test:
and triathletes training
muscle CA stores in LTT incremental Total dose 6 w 268.8 g EG = 9.7 vs. CG = 7.3; p > 0.05
M = 27 CG: PL +
endurance-trained cycle ergometer EG: BA 6.4 g/day (4 RPE (6–20) post test:
Chung et al. [35] (EG = 14; CG = training ↔
athletes, and whether (from 50 W, ≥60 rpm doses of 1.6 g, with EG = 19.0 vs. CG = 18.8; p > 0.05
13) D:
CA loading can improve until exhaustion) every meal) HR (bpm) post test:
A: 30.9 ± 7.9 PO: LTT
their endurance CG: maltodextrin EG = 181 vs. CG = 180; p > 0.05
years SO: [La], RPE,
performance. The authors declared paresthesia:
HR
no
Table 1. Cont.
Performance
Author Objective Subjects Variables Test Supplementation Protocol Results
in PO
To increase PA: Healthy I:
Oral suppl of 10 w Time (s) post test:
skeletal-muscle CA and subjects EG: BA + training
Total dose 10 w: 224 g EG = 1130 vs. CG = 1125; p > 0.05
augment muscle M = 24 CG: PL + training TTT 250 KJ cycle
Nutrients 2020, 12, 2490
Cochran et al. [45] EG: BA 3.2 g/day (2 doses of VO2 max (mL O2 ·kg−1 ·min−1 ) post test: ↔
buffering capacity during (EG = 12; CG = D: ergometer
1.6 g, every 12 h) EG = 52.2 vs. CG = 55.4; p > 0.05
a 6 week sprint interval 12) PO: TTT
CG: dextrose The authors declared paresthesia: no
training intervention. A: 22.5 ± 2.0 years SO: VO2 max
Oral suppl of 28 d
To assess if beta-alanine I:
Total dose 28 d: ~ 168 a 196 g Time (s) post test:
suppl could improve C/A: Rowers EG: BA + training
(80 mg·kg−1 ·d−1 ) EG = 391.0 vs. CG = 393.4; p > 0.05
2000 m M = 16 CG: PL + training TTT 2000 m
Ducker et al. [46] EG: BA ~ 6–7 g/day (4 doses [La] (mmol·L−1 ) post test: ↔
rowing-ergometer (EG = 7; CG = 9) D: rowing ergometer
of 1.5–1.75 g, with every EG = 12.5 vs. CG = 12.4; p > 0.05
performance in A: 26.0 ± 9.0 years PO: TTT
meal) The authors declared paresthesia: no
well-trained male rowers. SO: [La]
CG: glucose
To investigate the effect I: Oral suppl of 28 d
N/T: Healthy Time (s) post test:
of BA suppl on exercise EG: BA + training Total dose 28 d: 67.2 g
subjects LTT at 70% EG = 876 vs. CG = 522; p > 0.05
endurance and executive CG: PL + training EG: BA 2.4 g/day (3 doses of
Furst et al. [49] M = 8; F = 4 VO2 peak cycle [La] (mmol·L−1 ) post test: ↔
function in a D: 800 mg, with every meal)
(EG =7; CG = 5) ergometer EG = 6.6 vs. CG = 4.2; p > 0.05
middle-aged human PO: LTT CG: microcrystalline
A: 60.5 ± 8.6 years The authors declared paresthesia: no
population. SO: [La] cellulose
86
Time (s) post test:
To assess the effects of RA: Healthy I:
LTT incremental Oral suppl of 6 w EG = 992.4 vs. CG = 926.5; p: < 0.05
alanine suppl on VO2max , college students EG: BA + training
cycle ergometer Total dose 6 w: 84 g VO2 max (L O2 ·min−1 ) post test:
time to exhaustion, and M = 39 CG: PL + training
Ghiasvand et al. [50] (from 30 W, ≥70 EG: BA 2 g/day (5 doses of EG = 2.79 vs. CG = 2.81; p < 0.05 ↑
lactate concentrations in (EG = 20; CG = D:
rpm to 400 mg, with every meal) [La] (mg·dL−1 ) post test:
physical education male 19) PO: LTT
exhaustion) CG: dextrose EG = 27.9 vs. CG = 36.0; p < 0.05
students. A: 21.5 ± 1.1 years SO: VO2 max, [La]
The authors declared paresthesia: no
Oral suppl of 30 d
I:
To determine the effect of C/A: Aerobically Total dose 30 d: 159 g Time (s) post test:
EG: BA + training
30 days of BA suppl on fit males EG: BA 3 g/day for 7 d (2 EG = 1304 vs. CG = 1125; p > 0.05
CG: PL + training LTT cycle
Greer et al. [51] peak aerobic power and M = 14 doses of 1.5 g, every 12 h) + VO2 max (L O2 ·min−1 ) post test: ↔
D: ergometer
ventilatory threshold in (EG = 7; CG = 7) BA 6 g/day for 23 d (4 doses EG = 4.14 vs. CG = 3.97; p > 0.05
PO: LTT
aerobically fit males. A: 28.8 ± 9.8 years of 1.5 g, with every meal) The authors declared paresthesia: yes
SO: VO2 max
CG: maltodextrin
I: Time (s) post test:
To examine the effect of C/A: Rowers Oral suppl of 30 d
EG: BA + training EG = 410.3 vs. CG = 416.4; BA
BA only and BA with M = 20 Total dose 30 d: 192 g
CG: PL + training TTT 2000 m probability on PL: 96% effect (+)
Hobson et al. [47] sodium bicarbonate (EG = 10; CG = EG: BA 6.4 g/day (4 doses of ↑
D: rowing ergometer [La] (mmol·L−1 ) post test:
suppl on 2000 m rowing 10) 1.6 g, every 3–4 h)
PO: TTT EG = 14.7 vs. CG = 14.5; p > 0.05
performance. A: 23.0 ± 4.0 years CG: maltodextrin
SO: [La] The authors declared paresthesia: no
Table 1. Cont.
Protocol in PO
To examine the short-term
and chronic effects of BA I: Oral suppl of 28 d
suppl with and without FA: Healthy Total dose 28 d: ~ 170.8
Nutrients 2020, 12, 2490
EG: BA +
LTT incremental Time (s) post test:
creatine monohydrate on college students training g (0.1 g·kg−1 ·d−1 )
cycle ergometer EG = 1293 vs. CG = 1083; p > 0.05
body composition, aerobic, F = 15 CG: PL + EG: BA ~ 6.1 g/day
Kresta et al. [5] (from 50 W, ≥70 VO2 max (mL O2 ·kg−1 ·min−1 ) post test: ↔
and anaerobic exercise (EG = 8; CG = 7) training (around 4 doses of 800
rpm to EG = 41.53 vs. CG = 37.90; p > 0.05
performance and muscle CA A: 21.5 ± 2.8 D: mg, every 4 h)
exhaustion) The authors declared paresthesia: no
and creatine levels in years PO: LTT CG: dextrose and
college-aged recreationally SO: VO2 max maltodextrin
active females.
I:
To evaluate the cumulative S/E: Untrained Oral suppl of 8 w
EG: BA +
effect of resistance training collegiate Total dose 8 w: 108.8 g Time (s) post test:
training
and BA suppl on aerobic and females (32 doses) EG = 629.1 vs. CG = 591.1; p > 0.05
Outlaw et al. CG: PL +
anaerobic performance F = 15 LTT treadmill EG: BA 3.4 g/day (1 VO2 max (mL O2 ·kg−1 ·min−1 ) post test: ↔
[52] training
markers, as well as body (EG = 7; CG = 8) single dose before EG = 41.2 vs. CG = 38.6; p > 0.05
D:
composition, in collegiate A: 21.0 ± 2.2 training) The authors declared paresthesia: no
PO: LTT
females. years CG: maltodextrin
SO: VO2 max
87
I:
PA: Healthy EG: BA + Oral suppl of 23 d
To investigate the effects of Time (s) post test:
subjects training Total dose 23 d: 115 g
BA suppl on a 10 km running EG = 3210 vs. CG = 3480; p < 0.05
Santana et al. M = 16 CG: PL + TTT 10 km EG: BA 5 g/day (around
time trial and lactate [La] (mmol·L−1 ) post test: ↑
[48] (EG = 8; CG = 8) training treadmill 3 doses of 1.6 g, every 3
concentration in physically EG = 6.8 vs. CG = 10.8; p < 0.05
A: 29.4 ± 3.9 D: h)
active adults. The authors declared paresthesia: no
years PO: TTT CG: resistant starch
SO: [La]
I:
RA: Healthy
EG: BA + Oral suppl of 28 d
women Time (s) post test:
training LTT incremental Total dose 28 d: 134.4 g
To evaluate the effects of 28 F = 24 EG = 405 vs. CG = 388; p > 0.05
CG: PL + in treadmill EG: BA 4.8 g/day (3
Smith et al. [22] days of BA suppl on markers (EG = 13; CG = VO2 max (L O2 ·min−1 ) post test: ↔
training (from 10 km·h−1 doses of 1.6 g, in
of oxidative stress. 11) EG = 2.71 vs. CG = 2.64; p > 0.05
D: to exhaustion) intervals)
A: 21.7 ± 2.1 The authors declared paresthesia: yes
PO: LTT CG: maltodextrin
years
SO: VO2 max
Table 1. Cont.
Performance
Author Objective Subjects Variables Test Supplementation Protocol Results
in PO
Women
Time (s) post test:
I:
RA: Healthy EG = 313.8 vs. CG = 240.5; p > 0.05
Nutrients 2020, 12, 2490
To evaluate the effects of EG: BA +

subjects Oral suppl of 28 d [La] (mmol·L−1 ) post test:
BA suppl on training
M and F = 50 Total dose 28 d: 134.4 g EG = 13.15 vs. CG = 13.8; p > 0.05
high-intensity running CG: PL + LTT at 90% Vmax
Smith-Ryan et al. [21] (EG = 26; CG = EG: BA 4.8 g/day (3 doses of 1.6 Men ↔
performance and critical training treadmill
24) g, in intervals) Time (s) post test:
velocity anaerobic D:
A: 21.9 ± 2.7 CG: maltodextrin EG = 317.0 vs. CG = 322.6; p > 0.05
running capacity. PO: LTT
years [La] (mmol·L−1 ) post test:
SO: [La]
EG = 15.7 vs. CG = 13.8; p > 0.05
The authors declared paresthesia: yes
I:
FA: Healthy
EG: BA +
To determine the effect subjects LTT incremental Oral suppl of 28 d Time (s) post test:
training
of 28 days of BA suppl M and F = 30 cycle ergometer Total dose 28 d: 1792 g EG = 690.5 vs. CG = 703.6; p > 0.05
CG: PL +
Smith-Ryan et al. [53] on work physical (EG = 15; CG = (from 20 W, ≥60 EG: BA 6.4 g/day (4 dose of 1.6 VO2 max (mL O2 ·kg−1 ·min−1 ) post test: ↔
training
capacity test in heart 15) rpm until g, every 3–4 h) EG = 39.1 vs. CG = 43.4; p > 0.05 The
D:
rate threshold. A: 21.0 ± 2.1 exhaustion) CG: maltodextrin authors declared paresthesia: no
PO: LTT
years
SO: VO2 max
88
Acute effect of BA supplementation on aerobic–anaerobic transition zone
Time (s):
I:
EG = 366.5 vs. CG = 326.0; p < 0.05
EG: BA +
Distance (m):
C/A: High-level training Oral suppl
To determine the acute TTE (LTT and EG = 1828.6 vs. CG = 1651.4; p > 0.05
athletes CG: PL + Total dose: 30 mg·kg−1 body
effect of BA suppl on a LDT) at [La] (mmol·L−1 ):
M and F = 7 training mass Time ↑
Huerta et al. [17] limited time test at maximum EG = 14.80 vs. CG = 13.84; p > 0.05
(EG = 7; CG = 7) D: EG: BA from 1.5–2.1 g/day 60 Distance ↔
maximum aerobic speed aerobic speed in RPE (1–10):
A: 24.2 ± 4.4 PO: TTE (LDT min before TTE (LDT and LTT)
on endurance athletes. athletic track EG = 8.28 vs. CG = 7.60; p > 0.05
years and LTT) CG: simple carbohydrates
HR (bpm):
SO: [La], RPE,
EG = 185.4 vs. CG = 178.8; p > 0.05
HR
The authors declared paresthesia: no
A: age; BA: beta-alanine; bpm: beat per minute; C/A: competitive athlete; CA: carnosine; CG: control group; d: days; D: dependent variable; e/: every; EG: experimental group; EG1 :
experimental group 1; EG2 : experimental group 2; g/day: grams per day; g: grams; h: hours; HR: heart rate; I: independent variable; kg: kilograms; KJ: Kilo Joule; km: kilometers; L:
liters; LDT: limited distance test; LTT: limited time test; M: male; F: female; m: meters; mg: milligrams; mg·dL−1 : milligrams per deciliter; mg·kg−1 : milligrams per kilogram; min:
minutes; mmol·L−1 : millimole per liter; N/T: no training; PA: physically active; PL: placebo; PO: primary outcome; RA: recreational athlete; RPE; ratings of perceived exertion; s: seconds;
SO: secondary outcome; suppl: supplementation; T: time; TTE: time to exhaustion; TTT: time trial test; Vmax: maximum velocity; VO2 : oxygen uptake; VO2 max: maximal oxygen
uptake; vs: versus; VT: ventilatory threshold; W: watt; w: weeks; [La]: blood lactate concentration; p < 0.05: significant change; p > 0.05: non-significant change; ~: approximate; ↑:
positive effect; ↔: no effect.
Nutrients 2020, 12, 2490
Figure 5. Forest plot comparing the effects of BA supplementation on Time Trial Tests. BA: beta-alanine;
PL: placebo.
Figure 6. Forest plot comparing the effect of BA on Limited Time Test. BA: beta-alanine; PL: placebo.
3.4. Effect of BA on the Limited Distance Test

Two studies were considered for this analysis [17,28]. However, the study of Baesley et al. [28]
included two experimental groups for the LDT in their research design (30 min on a rowing ergometer
with 2.4 g/day of BA supplementation every 24 h and 30 min on a rowing ergometer with 4.8 g/day of
BA supplementation every 48 h). In this way, three studies were included in the meta-analysis that
calculated the effect of BA supplementation on the TTE test. Figure 7 shows that BA supplementation
generates a large and non-significant effect on distance in the TTE test (SMD, 4.27; 95% CI −0.25–8.79;
p = 0.06). The meta-analysis showed high heterogeneity among the studies reviewed (I2 = 94%;
p = 0.00001). All studies analyzed declared a beneficial effect of supplementation with BA on physical
performance in LDT [17,28].
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Nutrients 2020, 12, 2490
Figure 7. Forest plot comparing the effect of BA on Limited Distance Test. BA: beta-alanine; PL: placebo.
3.5. Effect of BA Supplementation on Secondary Outcomes

Of the total of 19 studies included in the systematic review and meta-analysis, 17 of them reported
on different parameters of physical performance. These parameters were defined as secondary
outcomes and included blood lactate concentration ([La]), VO2 max, RPE, and HR [32].
The meta-analysis of [La] (mmol·L−1 ) included 11 studies [5,17,21,28,29,35,43,46–49]. The total
number of cases supplemented with BA included 128 participants, while 121 participants were
supplemented with PL. The meta-analysis showed that BA supplementation generated a trivial and
non-significant effect on [La] post effort (SMD, 0.16; 95% CI −0.35–0.67; p = 0.53), while moderate
heterogeneity was present among the reviewed studies (I2 = 71%; p = 0.0001). A meta-analysis of
absolute VO2 max (LO2 ·min−1 ) included nine studies [5,22,28,43,45,50–53]. The total number of cases
supplemented with BA included 109 participants, while the PL group comprised 104 participants.
The meta-analysis showed that BA supplementation generated a trivial and non-significant effect
on absolute VO2 max (SMD, 0.17; 95% CI −0.11–0.45; p = 0.24), and low heterogeneity was observed
among the studies (I2 = 6%; p = 0.39). The meta-analysis of RPE [36] included four studies [17,28,35,43]:
the total number of cases supplemented with BA included 48 participants, while those supplemented
with PL comprised 46. The meta-analysis showed that BA supplementation generated a trivial
and non-significant effect on RPE (SMD, 0.03; 95% CI −0.52–0.58; p = 0.92), and low heterogeneity
was observed in the studies (I2 = 42%; p = 0.14). Finally, the meta-analysis for HR included three
studies [17,28,35], and the total of number of cases supplemented with BA included 39 participants,
while those supplemented with PL comprised 38. The meta-analysis showed that BA supplementation
generates a small and non-significant effect on HR (SMD, 0.30; 95% CI −0.66 to −1.26; p = 0.54),
and a high heterogeneity was observed among the studies reviewed (I2 = 75%; p = 0.008).
3.6. Paresthesia
At the end of this review, out of the 19 studies included in the systematic review and meta-analysis,
four of them reported paresthesia [21,22,29,51] (Table 1).
4. Discussion
In connection with the studies included in the systematic review and meta-analysis, the results
showed that BA supplementation presents an ES ranging from a small (0.2–0.5) to a large magnitude
(>0.8) in aerobic–anaerobic transition zones. At the same time, the results showed that changes in
physical performance are associated with both acute and chronic BA supplementation, while the
administered doses ranged from 1.5−6.4 g/day in periods ranging from 1 h before physical tests (acute
supplementation) to 10 weeks with one or several doses during the day (chronic supplementation).
At the end of this review, several studies concluded that the increase in physical performance after
BA supplementation is due to an increase in muscular CA concentrations [21,42,53]. The ergogenic
effect that generates increased CA is associated with intracellular regulation of pH (buffer), an increase
in Calcium (Ca2+ ) sensitivity in type I and II muscle fibers, and an increase in Ca2+ /H+ ion exchange;
as a consequence, these events showed an increase in muscular contractility [1]. For this reason,
direct supplementation with CA has been studied with inconclusive results [14,54,55], since CA is
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Nutrients 2020, 12, 2490
degraded into BA and L-histidine in the stomach [5]. Specifically, the low effectiveness of direct
supplementation with CA is related to the fact that L-histidine has a larger presence in plasma than
BA [1]. Because of this, BA supplementation shows better results than CA supplementation [44,50].
At the end of this review, the only secondary effect reported and associated with BA
supplementation was paresthesia [21,22]. This is a sensation of flushing associated with an irritant
tingling in the ears, scalp, hands, and torso [23]. The process responsible for paresthesia is the release of
L-histidine to form CA [9,12,27]. Paresthesia is transitory and can be avoided by dosing and ingesting
BA in smaller portions throughout the day [9,12,27].
4.1. Effect of BA on the Time Trial Test and Time to Exhaustion Test
BA supplementation and the subsequent increase in CA could diminish H+ circulation and prevent
the drop in intracellular pH during high-intensity exercise [50]. In fact, CA has been described as the
main buffering substance of H+ at the muscular level [56]. Previous studies have stated that blood and
muscular acidosis limit muscular contractility, which would favor the onset of fatigue [17,29,47,50].
At the same time, due to an increase in Ca2+ sensitivity to type I fibers, it has been mentioned that BA
supplementation can improve muscular contractile properties, delaying fatigue onset [17,57].
As mentioned above, the performance increase in aerobic–anaerobic transition zones is associated
with greater availability of muscular CA [20,50]. This way, evidence has shown that prolonged BA
supplementation in doses ranging from 2.0–6.4 g/day for 4–10 weeks can increase CA concentrations
between 64–80% [9]. In connection with acute supplementation in aerobic–anaerobic transition zones,
evidence is scarce [17]. In this regard, Huerta et al. [17] performed supplementation with 30 mg·kg−1
body mass (1.5–2.1 g/day) of BA 60 min prior to a TTE test. These researchers obtained an average
increase of 40.5 s at the end of the study (p < 0.05). Despite that, and due to the limited evidence
relating acute supplementation with BA on physical performance in aerobic–anaerobic transition zones,
it is impossible to guarantee a real effect in this physiological zone. However, the increase in physical
performance observed in this review is supported by greater bioavailability of CA, an increase that is
observed shortly after the intake of BA [58]. This raises the possibility of studying the acute effects of
BA using different protocols and observing the real effects in aerobic–anaerobic transition zones.
The ES for distance on the TTE test was large (ES = 4.27), while TTT and time on the TTE test
was small (ES = −0.36 and 0.25, respectively). In light of these results, these last values show a small
effect of BA supplementation on physical performance in aerobic–anaerobic transition zones. However,
considering that an elite athlete’s performance is bound by extremely tight margins (probably difficult
to measure statistically), in real practice, a small ES could be of great importance, since it has been
proven that in world finals, differences lower than 3% can be found between first and last place [1].
4.2. Effect of BA on Secondary Outcomes

BA supplementation could prevent the drop in intracellular pH during high-intensity exercise (due
to an increase in muscular CA bioavailability) and, as a consequence, generate less lactate accumulation
with the same intensity of physical exercise [48,50]. Regarding lactate accumulation, it is important
to mention that this is not the cause of H+ accumulation, but a high intensity of exercise produces
a decrease in pH and an increase in intramuscular and blood [La] simultaneously, transforming lactate
in a good marker of physical effort [8]. Despite the theoretical background, the meta-analysis showed
a trivial effect on [La] post effort (ES = 0.16).
The influence of BA supplementation on aerobic performance has been widely studied [14,20,27,59];
however, the meta-analysis showed a trivial effect of BA on VO2 (ES = 0.17) [51,60]. Apparently,
the increase in VO2 is less dependent on the buffer qualities that BA supplementation produces [20]. It is
possible that the improvement in VO2 reported in some studies included in the meta-analysis is more
connected to physical training in aerobic–anaerobic transition zones than to BA supplementation [61,62].
In connection to RPE, some studies have shown a good correlation between RPE and HR
during physical exercise in healthy subjects (1 point of RPE equals approximately 10 bpm). More so,
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Nutrients 2020, 12, 2490
the metabolic thresholds have been associated with specific values on the Borg scale [36]. Likewise,
it has been shown that a lower value of RPE for the same workload entails a metabolic adaptation
after the training process [63]. Despite these lines of theoretical evidence, the studies included in the
meta-analysis showed a trivial effect on RPE reported by the participants (ES = 0.03). This value can be
derived from the level of demand experienced by the participant; it is also possible that they exerted
themselves to the maximum effort in all tests, reaching the upper limits of the RPE scales used [36].
As a consequence improved cardiac contractility, it has been described that CA can increase
HR [53]. In addition, intracellular pH has proven to be a modulator of cardiac function, increasing the
entrance of Ca2+ during action potentials, facilitating cardiac contraction [64]. This information makes
it possible to anticipate an increase in HR after BA supplementation [53]. However, HR is dependent
on the intensity of physical effort; hence, if the participants exerted themselves to the maximum in all
tests, it is likely that post-effort HR values would not show major variations when supplementing with
BA (ES = 0.30).
Finally, due to a limited number of studies, only the secondary outcomes mentioned above were
used. Subdividing the 10 TTT studies and 11 TTL studies to perform a meta-analysis by gender,
age, exercise modalities, or physical activity level would have generated a bias in the information
obtained [38].
4.3. Limitations
The main limitations of this research were the access to information and unspecific data reported
by some studies included in the systematic review and meta-analysis. However, the limitations were
solved by contacting the authors of each study. Only one document was not included because no
answer was received. Another important limitation in this review was the limited number of studies
that used TDL as a primary outcome [17,28].
5. Conclusions
Both acute and chronic supplementation with BA in doses of 1.5–6.4 g/day showed a small and
non-significant effect on physical performance in aerobic–anaerobic transition zones. Physiologically,
this positive change is due to the buffer effects generated by the larger bioavailability of intracellular
CA, which allows for a delay in the onset of fatigue in the TTT and TTE tests within this specific
physiologic zone. That is why small changes in individual performance must be considered, since they
can be the difference between success and failure among high-level and elite athletes.
Furthermore, the findings showed evidence that acute supplementation with BA is scarce,
generating alternatives for researchers to study the effect of this form of supplementation with different
BA doses and formats on performance in aerobic–anaerobic transition zones.
6. Practical Applications
Coaches and athletes looking for an ergogenic aid to enhance physical performance in
aerobic–anaerobic transition zones should consider both acute and chronic supplementation with BA.
The dosage can range from 30 mg·kg−1 of body mass in acute supplementation to 6.4 g/day in chronic
supplementation. The latter may be administered in several doses per day. However, it is advisable to
check the dosage and supplementation formats with qualified professionals.
Finally, in order to avoid the presence of paresthesia after supplementation with BA, it is
recommended that BA be dosed and ingested in small portions throughout the day (the amount
suggested for these doses is 1.6 g of BA per dose) [9]. The second recommendation to avoid paresthesia
is to also ingest a large amount of carbohydrates 60 min before ingesting BA (the suggested carbohydrate
load is 2 g·kg−1 of body mass) [17].
Author Contributions: Á.H.O., C.T.C., and M.F.P.S.: conception, methodology, investigation, data curation,
writing—original draft preparation, writing—review and editing. G.B.-F.: visualization and writing—review and
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Nutrients 2020, 12, 2490
editing. C.J.A.: supervision and project administration. All authors have read and agreed to the published version
of the manuscript.
Funding: The authors declare no funding sources.
Conflicts of Interest: The authors declare no conflict of interests.
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Article
Can Creatine Supplementation Interfere with Muscle
Strength and Fatigue in Brazilian National Level
Paralympic Powerlifting?
Carlos Rodrigo Soares Freitas Sampaio 1 , Felipe J. Aidar 1,2,3,4, *, Alexandre R. P. Ferreira 5 ,
Jymmys Lopes dos Santos 6 , Anderson Carlos Marçal 1,3 , Dihogo Gama de Matos 1 ,
Raphael Fabrício de Souza 1,2 , Osvaldo Costa Moreira 7 , Ialuska Guerra 8 , José Fernandes Filho 9 ,
Lucas Soares Marcucci-Barbosa 10 , Albená Nunes-Silva 10 , Paulo Francisco de Almeida-Neto 11 ,
Breno Guilherme Araújo Tinoco Cabral 11 and Victor Machado Reis 12
1 Group of Studies and Research of Performance, Sport, Health and Paralympic Sports (GPEPS),
Federal University of Sergipe (UFS), São Cristovão 49100-000, Sergipe, Brazil;
[email protected] (C.R.S.F.S.); [email protected] (A.C.M.);
[email protected] (D.G.d.M.); [email protected] (R.F.d.S.)
2 Department of Physical Education, Federal University of Sergipe (UFS),
São Cristovão 49100-000, Sergipe, Brazil
3 Program of Physical Education, Federal University of Sergipe (UFS), São Cristovão 49100-000, Sergipe, Brazil
4 Program of Physiological Science, Federal University of Sergipe (UFS),
São Cristovão 49100-000, Sergipe, Brazil
5 College of Physical Education and Exercise Science, University of Brasília (UnB), Brasília 70910-900, Brazil;
[email protected]
6 Program in Biotechnology, Northeast Network in Biotechnology (RENORBIO), Federal University of
Sergipe (UFS), São Cristovão 49100-000, Sergipe, Brazil; [email protected]
7 Institute of Biological Sciences and Health, Federal University of Viçosa, Campus Florestal,
Minas Gerais 35690-000, Brazil; [email protected]
8 Federal Institute of Education, Science and Technology of Ceará (IFCE), Campus of Juazeiro do Norte,
Ceará 63040-540, Brazil; [email protected]
9 Brazilian Paralympic Academy, Brazilian Paralympic Committee, São Paulo 04329-000, SP, Brazil;
jff[email protected]
10 Laboratory of Inflammation and Exercise Immunology, Sports Center, Physical Education Scholl,
Federal University of OuroPreto (UFOP), OuroPreto, Minas Gerais 35400-000, Brazil;
[email protected] (L.S.M.-B.); [email protected] (A.N.-S.)
11 Department of Physical Education, Federal University of Rio Grande do Norte (UFRN), Natal, Rio Grande
do Norte 59078-970, Brazil; [email protected] (P.F.d.A.-N.); [email protected] (B.G.A.T.C.)
12 Research Center in Sports Sciences, Health Sciences and Human Development (CIDESD), Trásos Montes and
Alto Douro University, 5001-801 Vila Real, Portugal; [email protected]
Abstract: The aim of the present study was to analyze the effect of creatine (Cr) supplementation on
peak torque (PT) and fatigue rate in Paralympic weightlifting athletes. Eight Paralympic powerlifting
athletes participated in the study, with 25.40 ± 3.30 years and 70.30 ± 12.15 kg. The measurements of
muscle strength, fatigue index (FI), peak torque (PT), force (kgf), force (N), rate of force development
(RFD), and time to maximum isometric force (time) were determined by a Musclelab load cell.
The study was performed in a single-blind manner, with subjects conducting the experiments first
with placebo supplementation and then, following a 7-day washout period, beginning the same
protocol with creatine supplementation for 7 days. This sequence was chosen because of the lengthy
washout of creatine. Regarding the comparison between conditions, Cr supplementation did not
show effects on the variables of muscle force, peak torque, RFD, and time to maximum isometric force
(p > 0.05). However, when comparing the results of the moments with the use of Cr and placebo,
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a difference was observed for the FI after seven days (U3 : 1.12; 95% CI: (0.03, 2.27); p = 0.02); therefore,
the FI was higher for placebo. Creatine supplementation has a positive effect on the performance of
Paralympic powerlifting athletes, reducing fatigue index, and keeping the force levels as well as PT.
Keywords: Paralympic powerlifting; supplementation; creatine; performance
1. Introduction
Powerlifting (PL) is an international sport where competitors attempt to lift a maximum amount
of weight in threeprimary lifts: the bench press, the squat, and the deadlift. These threelifts provide
widely accepted measures of upper-body, lower-body, and total body strength [1–3]. At all levels of PL,
each competing athlete is ranked based on the best of the threevalid attempts afforded for the bench
press, squat, and deadlift [1–3]. The threeare then “totaled,” providing a measure of the total weight
lifted and determining each athlete’s overall place in the competition. The skillful execution of each of
the threeafforded lifts for the bench press, squat, and deadlift during competitions can be influenced
by the subjects’ strength and muscle fatigue [1–3].
In this sense, many of these athletes have used ergogenic aids to keep body conditioning, enhancing
recovery, and physiological adaptations during training programs and between competitions [3,4].
The efficacy of ergogenic has always attracted great attention, and numerous researchers have
sought to combine ergogenic and exercise training programs to reinforce the benefits of training.
Creatine (Cr) is a popular ergogenic aid among athletes at all levels [5,6]. Cr is a non-protein
nitrogenous compound—methyl-guanidine-acetic acid—composed of three amino acids (arginine,
glycine, and methionine). It is found mainly in skeletal muscle (95%) and plays an important role in
rapid energy provision during muscle contraction through the ATP-PCr system [7].
Cr supplementation tends to potentiate the effect of strength training that would promote
physiological responses and adaptations that positively interfere with the increase in muscle strength,
power, hypertrophy, and local muscle endurance [8]. On the other hand, observing variables related
to muscle recovery, there areindications that Cr supplementation could reduce muscle damage after
exercise via sarcolemma stabilizing mechanisms [9] and regulate mitochondrial permeability [10].
Studies have demonstrated the beneficial effects of Cr supplementation on performance following
resistance training [9–11].
Although the literature has shown the positive impacts of creatine supplementation [8–11],
there is a lack of strong evidence about the efficacy of creatine supplementation on elite powerlifting
athletes. More evidence is required to testing the efficacy of creatine to minimize fatigue index,
which might enhance muscle strength, considering the need for new approaches that contribute to
better performance.
Therefore, this was the first study to investigate the effects of creatine supplementation in elite
Paralympic powerlifting athletes. We hypothesized that creatine might affect positively the muscle
strength and reduce fatigue during high-intensity resistance training used in Paralympic powerlifting
training. Thus, the aim of the present study was to analyze the effects of Cr supplementation on
indicators of torque, force, time, and fatigue index in Paralympic powerlifting athletes.
2.1. Sample
The sample consisted of eight Paralympic powerlifting athletes participating in the extension
project of the Federal University of Sergipe Brazil. All participants were Brazilian level competitors,
eligible for the sport [12], and ranked among the ten best in their respective categories. Among the
deficiencies, two athletes presented spinal cord injury due to accidents with an injury below the eighth
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thoracic vertebra; two with sequelae due to polio; two had lower limb malformation (arthrogryposis);
two had cerebral palsy. The sampling power was calculated based on previous results of our studies [2,3],
with an effect size of 0.98 that combined with a standard of α < 0.05 and β = 0.80. Thus, it was possible
to estimate a sample power of 0.88, suggesting that the sample size has sufficient statistical strength to
answer the research approach.
The characterization of the sample is shown in Table 1.
Table 1. Characterization of subjects.
(Mean ± SD)
Age (years) 25.40 ± 3.30
Weight (Kg) 70.30 ± 12.15
Experience (years) 2.45 ± 0.21
1RM Adapted Bench press (Kg) 119.99 ± 12.14 *
1RM/weight 1.71 ± 0.27 **
* All athletes with loads that keep them in the top 10 of their categories nationwide. ** Values above 1.4 on bench
press would be considered elite athletes, according to Ball and Weidman. 1RM: 1 repetition maximum.
The athletes voluntarily participated in the study and signed a free and informed consent form in
accordance with Resolution 466/2012 of the National Commission for Research Ethics (CONEP) of the
National Health Council and the ethical principles of the latest version of the Declaration of Helsinki
(and the World Medical Association). The project was submitted to the Research Ethics Committee of
the Federal University of Sergipe and approved with the following opinion 2,637,882.
This study was carried out at the Federal University of Sergipe, from 09:00 h to 13:00 h,
and was developed in four weeks, the first one aimed at familiarization and testing of 1 repetition
maximum (1RM), force (force (Kgf) and force (N)), peak torque (PT), rate of force development (RFD),
time to maximum isometric force (time), and fatigue index (FI), according to the items Section 2.4.
Force Measurements and Section 2.5. Load Determination, respectively.
The experimental design of the study is provided in Figure 1.
Figure 1. Experimental design—weekly schedule of tests and washout. 1RM: 1 repetition maximum.
Training carried out three times a week, and the remaining days weredestined to rest [2].
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2.2. Instruments
Weighing of the athletes was performed on a digital platform-type Michetti (Micheletti, São Paulo,
SP, Brazil) electronic scale, with a maximum supported weight capacity of 3000 kg and a size of
1.50 × 1.50 m. For the bench press exercise, an official straight bench (Eleiko Sport AB, Halmstad,
Sweden), approved by the International Paralympic Committee [12], with a total length of 210 cm
was used. The IPC-approved powerlifting Olympic bar is serrated and has grooves in its material,
has a total length of 220 cm, weighing 20 kg. On the bar, there is a marking for the narrowest and
widest footprint, according to the International Paralympic Committee [12] official rules 2016–2017,
ranging from 42 cm to 81 cm.
2.3. Supplementation
We chose to use a single-blind method with a treatment order that was not counterbalanced
due to the lengthy washout time required for muscle creatine to return to pre-supplementation
values [13]. Therefore, initially, participants ingested 20 g maltodextrin (placebo, Max Titanium® ,
Supley, Matão, SP, Brazil), followed by 7 days of washout period. Subsequently, 20 g of creatine
monohydrate (Max Titanium® , Supley, Matão, SP, Brazil; 99.9% purity) was administered for another
7 days. The total daily amount of supplement was divided into four equal portions and consumed with
food throughout the day. Creatine and placebo were identical in taste, color, texture, and appearance.
2.4. Force Measurements

Measurementsof muscle strength, fatigue index (FI), peak torque (PT), force (Kgf), force (N),
rate of force development (RFD), and time to maximum isometric force (time) were determined by a
Musclelab load cell (Model PFMA 3010e MuscleLab System; Ergotest, Langesund, Norway), attached
to the adapted bench press, using 21 HN Simplex carabiners Spider HMS Simond (Simond, Chamonix,
France), approved for climbing by Union InternationaledesAssociations d’Alpinisme (UIAA). A steel chain
with a breaking load of 2300 kg was used to secure the load cell to the seat. The perpendicular distance
between the load cell and joint center was determined and used to calculate joint torques and fatigue
index [14].
Isometric peak torque (PT) was measured by the maximum torque generated by the upper limb
muscles. The PT was determined by the product of the isometric force peak, measured between the
load cell cable attachment point and the adapted bench press bench, which was adjusted so that an
elbow angle was close to 90◦ and at a distance 15 cm from the starting point (chest to bar), verified with
an apparatus for measuring the amplitude of angular movement, Model FL6010 (Sanny, São Bernardo
do Campo, SP, Brazil). Participants were instructed to perform a single maximal movement, seeking
elbow extension (as soon as possible) and relaxing for PT assessment.
For the fatigue index (FI) evaluation, the same exercise was performed, and the subjects determined
to maintain the maximum contraction for 10 s, where the index was determined by dividing the initial
PT in relation to the final PT, subtracted from one. FI = ((final PT-initial PT/final PT) × 100). Thus, the
results in Newton (N) were conceived by the formula N = (M) × (C) × (H), where M = body mass in
Kg, C = 9.81 (m·s−2 ), H = bar height relative to load (45.0 cm), corresponding to the height at which
the equipment was fixed, adopting an angle of the forearm with the arm of 90◦ , adapted from the
methodology from Milner-Brown et al. [15].
2.5. Load Determination

To determine the training load, the 1RM test was performed, on the adapted bench press [12],
where each subject started the attempts with a weight that he believed could be lifted only once using
the maximum effort. Weight increments were then added until the maximum load that could be lifted
once was reached. If the practitioner could not perform a single repetition, 2.4 to 2.5% of the load used
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in the test was subtracted [16]. The subjects rested for 3–5 min between the attempts. The 1RM test
was performed within two weeks at least 72 h prior to the intervention.
For the PT, force, RFD, time, and FI tests, three attempts were made in the PT test, where subjects
were evaluated with the bar at 15 cm from the chest, and with an elbow angle of 90◦ , where they made
the greatest force possible once, and this procedure was repeated three times after a rest of five minutes
between the attempts. For the evaluation of the FI, the subjects remained to dothe maximum isometric
contraction for one minute, with the bar 15 cm from the chest, and the loss of PT was verified between
the 10 s and the initial moment of the test. The 10 s time was adopted in view of that shown in a
study with Paralympic powerlifting [17], where the execution of 1RM, the target of the competition,
would not amount to more than 10 s. All subjects underwent the test before and after training with a
minimum interval of 10 min between the tests and the training session [18].
2.6. Intervention
The intervention protocol consisted of warm-up for upper limbs, using three exercises (abduction
of the shoulders with dumbbells, elbow extension in the pulley, and rotation of the shoulders with
dumbbells) with three sets of 10 to 20 repetitions [19]. Soon after, a specific warm-up was performed
on the bench press with a 30% load of 1RM, 10 slow repetitions (3:1 s, eccentric:concentric), and 10 fast
repetitions (1:1 s, eccentric:concentric),followed with five sets of bench press of five maximum
repetitions (5 sets–85 at 90% RM with 3–5 min of rest), using a fixed load. During the test, athletes
received verbal encouragement in order to achieve maximum performance [19]. To perform the bench
press, an official straight bench (Eleiko Sport AB, Halmstad, Sweden), approved by the International
Paralympic Committee, was used [12].
2.7. Statistic
The normality of the data was verified by the Shapiro Wilk and Z-score tests for asymmetry and
kurtosis (−1.96 to 1.96). The assumption of normality was denied, and subsequently, the transformation
of the data by the square root (i.e., from non-parametric to parametric) was not successful,
and subsequently, the attempt to the logarithmic transformation of the data by the log on the
basis of 10 was also unsuccessful. In this sense, comparisons between the medians of the same
intervention (creatine × creatine; placebo × placebo) in the different conditions of the study (before,
after training, after 7 days) were performed using the Kruskal–Wallis test. When differences were found,
the Mann–Whitney U test was used to identify the different data set and, subsequently, Bonferroni
correction. The differences between interventions (creatine × placebo) in the different conditions of
the study (before, after training, after 7 days) were analyzed by the Mann–Whitney U test. The effect
size between the median differences and their respective 95% confidence intervals was analyzed
using the Cohen’s U3 index test, so the magnitude used was the one proposed by Espirito Santo and
Daniel [20]: insignificant: <0.19; small: 0.20–0.49; average: 0.50–0.79; large: 0.80–1.29; very large: <1.30.
All analyses were performed using open-source software R (version 3.6.2, R Foundation for Statistical
Computing, Vienna, Austria), considering the significance of p < 0.05.
3. Results
Figure 2 shows the results of the effect of creatine supplementation (intra-conditions) in relation
to the variables studied.
Regarding the use of creatine, Figure 2 shows that, for the RFD variable, significant differences
were identified in the after training and after 7 days conditions in relation to the before condition
(U3 = 1.33; CI 95%: [0.15]–[2.52]; p = 0.02), while for the use of placebo, there was no significant
difference. Regarding thetime to maximum isometric force, there was a difference during the use
of creatine for the after training condition compared to the before condition (U3 = 1.54; CI 95%:
[0.32]–[2.76]; p = 0.01). In relation to the placebo, there was a significant difference in the conditions
after training and after 7 days in relation to the before condition for the variable time to maximum
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isometric force (U3 = 0.76; CI 95%: [−0.34]–[1.87]; p = 0.04). There were significant differences in
the after training condition in relation to the before and after 7 days conditions, and from the after
7 days condition to the before condition when using creatine for the fatigue index (U3 = 7.97; CI 95%:
[4.76]–[11.1]; p = 0.0009). In relation to the use of placebo, significant differences were found in the
conditions after training and after 7 days in relation to the before condition for the fatigue index
(U3 = −12.9; CI 95%: [−17.9]–[−7.91]; p = 0.04).
Figure 2. Intra-condition comparisons regarding force indicator at different times. Kgf = Kilograms
force. N = Newtons. Nm = Nanometer. p = Value of the degree of statistical significance. m/s = Meters
per Second. % = Percentage. * = Significant differences for the before condition. § = Significant
difference for the after 7 days condition. p = Value of the degree of statistical significance.
Table 2 reports that the only statistical difference between interventions with the use of creatine
and placebo was in the fatigue index (%) in the condition after 7 days (U3 : 1.12; 95% CI: [−0.03]–[2.27];
p = 0.02), where the fatigue index (%) was higher for the intervention using the placebo.
Table 2. Comparison of moments using creatine and using placebo in the different conditions of
the study.
Before After Training After 7 Days

Tests Creatine Placebo Creatine Placebo Creatine Placebo
MD IIQ MD IIQ MD IIQ MD IIQ MD IIQ MD IIQ
Force (Kgf) 96.4 1.50 92.1 14.6 95.5 9.00 89.8 23.1 99.6 16.60 94.9 11.0
Force (N) 945.2 122.1 902.5 143.4 935.9 109.5 880.6 225.1 976.2 161.2 930.0 162.8
Peak torque (Nm) 425.3 54.9 406.1 64.5 421.1 48.9 396.2 101.6 439.3 73.30 418.5 73.4
Rate of force development 629.0 233.3 674.2 331.8 1.137 472.5 956.5 595.8 1.239 578.5 845.0 513.2
Time (m/s) 0.708 0.317 0.433 0.622 1.000 0.205 1.130 0.850 0.950 0.275 0.987 0.170
Fatigue index (%) 21.9 8.80 24.7 4.1 72.1 4.80 76.1 7.0 66.2 14.70 77.9 * 11.8
MD = Median; IIQ = Interquartile range. Kgf = Kilo grams force. N = Newtons. Nm = Nanometer. m/s = Meters
per Second. % = Percentage, Time = Time to maximum isometric force. * = Significant statistical difference p = 0.02.
p = Value of the degree of statistical significance.
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Figure 3 shows graphically the behavior of the peak torque (Nm) and the percentage of the fatigue
index (%) during the moments of the study (before, after training, and after 7 days), showing that
in relation to the peak torque (Nm), the behavior was similar for creatine and placebo conditions.
Whereas for the fatigue index (%), at the moment after 7 days, a lower percentage was demonstrated in
the creatine condition in relation to the placebo condition.
Figure 3. The behavior of peak torque (Nm) and fatigue index (%) at different times.
4. Discussion
The objective of the present study was to analyze the effects of Cr supplementation on the
indicators of torque, force, and muscle fatigue in athletes of Paralympic powerlifting. The main results
were: (1) Cr supplementation did not show effects on the variables of muscle strength, peak torque,
RFD, and time to maximum isometric force. (2) Cr supplementation reduced FIafter 7 days of use.
In the present study, when comparing Cr results with placebo, no significant differences were
identified in relation to variables related to muscle strength. In this context, Zuniga et al. [21] examined
the effects of 7 days of Cr supplementation on the strength of upper and lower limbs of 22 men and
concluded that there was no statistically significant difference between the placebo condition and the
Cr condition in all the muscle strength variables analyzed. In addition, Hamilton et al. [22] concluded
that Cr supplementation combined with resistance training when relative loads and volumes were
the same as a placebo condition did not result in a training advantage in absolute or relative strength
performance. Syrotuik et al. [23] did not observe significant differences in the strength of the upper
limbs when comparing two conditions after an intervention with Cr and placebo for 5 days.
Buts et al. [24] showed in a systematic review the scientific information from the years 1980 to 2017,
stating that the data on the improvement of sports performance through creatinewere inconsistent.
In addition, a meta-analysis made up of 100 different studies has shown that in the short term,
Cr supplementation does not have significant effects on specific sports performance [25]. Moreover,
in previous studies, Cr supplementation, in the short term, hasnot demonstratedeffects on specific
strength levels related to different sports [26,27].
The results of the present research also showed that FIof the supplemented condition was
approximately 16% lower when compared to placebo. In contradiction to the present study,
Bazzucchiet et al. [28] evaluated 16 trained men with daily supplementation of 5 g of Cr + 15 g
of maltodextrin or 20 g of maltodextrin and evaluated the maximum voluntary isometric contraction
and dynamic contractions and fatigue for the flexor muscles of the elbow. The authors concluded
that creatinecouldimprove neuromuscular function during voluntary contractions. In addition, they
indicated that, according to the electromyography analysis, no significant differences were found
between the conditions regarding muscle fatigue.
Possible explanations for the improvement of the Cr condition’s FI can be speculated, in addition
to neuromuscular adaptation, by the increase in glycogen storage. An increase in glucose transporter
type 4 (GLUT4) expression is suggested when Cr supplementation is combined with exercise [29];
positive effects of Cr supplementation are seen at initial levels and also by maintaining high levels
of muscle glycogen for up to 2 h [30]. In addition, training tends to promote central and peripheral
fatigue, along with other endocrine, immunological, inflammatory, and oxidative stress [31]. Moreover,
training tends to modulate physiological adaptation and improve physical performance indicators [32].
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In this context, supplementation can be used as a nutritional strategy for athletes to improve their
physiological adaptation and performance [33].
Resistance to fatigue and the ability of the muscle to regenerate during intermittent high-intensity
exercise are important qualities of neuromuscular function [34]. In addition, Cr can help protect against
injury and muscle damage induced by strenuous contractile activities [35]. In athletes, who performed
ultra-resistance tests, supplemented with 20 g/d of maltodextrin plus 50 g of Cr for 5 precompetitive
days, decreased plasma creatine kinase activities, lactate dehydrogenase, preventing the increase in
plasma oxaloacetic glutamic acid and glutamic pyruvic acid, activities have been observed [36].
In subjects with spinal cord injury, Cr levels improve muscle strength parameters, and this has a
positive effect on the performance of daily activities and body health [37]. In Paralympic weightlifting
athletes, who have suffered spinal cord injury, creatine can help to maximize the performance of the
upper limbs by reducing the FI and providing a faster recovery during the practices provided by the
sport [38].
In addition, it has been shown in the literature that creatine supplementation appears to reduce the
spread of secondary injuries and improves the quality of the neuromotor system’s fitness [39]. However,
caution is recommended regarding the water balance during the consumption of the supplement [24].
However, despite the relevance of the results, the present study had some limitations:
(1) The evaluation was done in an acute way. (2) The improvement in FI might have been a
result of the Cr supplementation time phase, that is, due to the fact of the time/order effect in which
the study was carried out. Cr supplementation was performed in a second moment, and because of
that, there might have been an adaptation to training, which might have influenced the athletes’ FI
reduction. (3) Athletes’ diets were not changed during the study. Therefore, new studies should be
carried out with a long washout period as well as other research designs.
5. Conclusions
It was concluded that creatine supplementation has a positive effect on the performance of
elite Paralympic powerlifting athletes, reducing fatigue in the execution of the exercise, and keeping
theforce levels.
Author Contributions: Conceptualization, F.J.A., R.F.d.S., O.C.M., I.G., L.S.M.-B., A.N.-S., P.F.d.A.-N., B.G.A.T.C.
and V.M.R.; Data curation, A.R.P.F., J.L.d.S. and D.G.d.M.; Formal analysis, J.L.d.S. and A.C.M.; Investigation,
C.R.S.F.S., F.J.A., R.F.d.S. and I.G.; Methodology, C.R.S.F.S., F.J.A., A.C.M., A.N.-S., P.F.d.A.-N., B.G.A.T.C. and
V.M.R.; Project administration, A.C.M. and J.F.F.; Writing—original draft, F.J.A., J.L.d.S., D.G.d.M., R.F.d.S.,
O.C.M., I.G., J.F.F. and V.M.R.; Writing—review & editing, A.R.P.F., D.G.d.M., J.F.F., L.S.M.-B., A.N.-S., P.F.d.A.-N.,
B.G.A.T.C. and V.M.R. All authors have read and agreed to the published version of the manuscript.
Funding: This work received funding from FCT—Fundação para a Ciência e Tecnologia (UID04045/2020).
We thank the support of the Foundation for Support to Research and Technological Innovation of the State of
Sergipe-FAPITEC/SE for granting research and to Brazilian National Council for Scientific and Technological
Development (CNPq).
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nutrients
Review
Effects of Dietary Nitrates on Time Trial Performance
in Athletes with Different Training Status:
Systematic Review
Tomáš Hlinský *, Michal Kumstát and Petr Vajda
Faculty of Sports Studies, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic;
[email protected] (M.K.); [email protected] (P.V.)
Received: 25 August 2020; Accepted: 4 September 2020; Published: 8 September 2020
Abstract: Much research has been done in sports nutrition in recent years as the demand for
performance-enhancing substances increases. Higher intake of nitrates from the diet can increase the
bioavailability of nitric oxide (NO) via the nitrate–nitrite–NO pathway. Nevertheless, the increased
availability of NO does not always lead to improved performance in some individuals. This review
aims to evaluate the relationship between the athlete’s training status and the change in time trial
performance after increased dietary nitrate intake. Articles indexed by Scopus and PubMed published
from 2015 to 2019 were reviewed. Thirteen articles met the eligibility criteria: clinical trial studies
on healthy participants with different training status (according to VO2max ), conducting time trial
tests after dietary nitrate supplementation. The PRISMA guidelines were followed to process the
review. We found a statistically significant relationship between VO2max and ergogenicity in time
trial performance using one-way ANOVA (p = 0.001) in less-trained athletes (VO2 < 55 mL/kg/min).
A strong positive correlation was observed in experimental situations using a chronic supplementation
protocol but not in acute protocol situations. In the context of our results and recent histological
observations of muscle fibres, there might be a fibre-type specific role in nitric oxide production and,
therefore, supplement of ergogenicity.
Keywords: nitric oxide; dietary supplements; oxygen consumption; muscle fibres; physical activity
1. Introduction
Nitric oxide (NO) plays a crucial role in signalling and physiological regulatory functions
of the human body which are crucial to exercise economy and performance (i.e., vasodilatation,
mitochondrial respiration, glucose and calcium (Ca2+ ) homeostasis, skeletal muscle contractility and
fatigue development). Because the NO molecule is highly unstable, there is a constant need for its
regeneration [1–4].
Interestingly the substrates for NO syntheses (L-arginine and nitrates) come from our daily diet
and therefore can be manipulated [5]. Increased availability of NO via diet has been linked to ergogenic
effects [6,7]. However, not all athletes can benefit from these nutritional strategies [8,9]. It seems the
effectiveness of NO inducing foods and supplements is limited by an athlete’s training status (aerobic
fitness) and muscle fibre type ratio [10].
1.1. NO Metabolism and Physiological Importance

Nitric oxide synthesis in the human body is carried out via two pathways: the NO synthase
(NOS)-dependent pathway and nitrate–nitrite–NO (NO3 − –NO2 − –NO) pathway [11]. Formation of
NO via the NOS-dependent pathway is carried out through the utilisation of L-arginine and oxygen
(O2 ). Thus, this reaction relies on the delivery of O2 [12]. Insufficient O2 delivery during high-intensity
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exercise may cause this pathway to become dysfunctional [13]. Therefore, the O2 -independent pathway
can substitute NO production [14] via the reduction of NO3 − from the diet (e.g., green leafy vegetables,
beetroot, radish) [15]. Nitrates are reduced by oral anaerobic bacteria to NO2 − [16]. Subsequently, part of
the total NO2 − is reduced to NO in the acidic environment of the stomach where it protects the organism
from some pathogens [17]. The rest of the NO2 − is then transported via the upper gastrointestinal tract
into the blood reaching its plasma peak level 2–3 h postprandially [18]. The reduction of NO2 − to NO
substitutes the O2 -dependent NOS pathway in various tissues under hypoxic or acidic conditions [19].
These are conditions that typically occur in working muscles during vigorous exercise [20].
Increased NO bioavailability via food or supplements may increase exercise performance, as it is
the critical factor in three physiological mechanisms related to physical exercise. Firstly, NO increases
skeletal muscle O2 delivery via vasodilatation [21]. Secondly, it reduces the O2 cost of mitochondrial
ATP resynthesis via an increased number of ATP molecules (P) formed per O2 molecules consumed
(O) in the electron transport chain (P/O ratio) [22], possibly via the interaction with five-coordinated
cytochrome C oxidase [23,24]. Lastly, it improves the efficiency of muscle contractility via reduced
creatine phosphate (PCr) cost of force production [25] and changes in Ca2+ metabolism within the
muscle cells [26]. Enhancing these physiological mechanisms leads to more efficient energy metabolism,
lower O2 demands of the working muscles and higher muscle contractility [26] and therefore an
increase in exercise economy and performance [1,22,25].
1.2. The Role of Dietary Nitrates in Exercise Physiology

Dietary nitrates (DN) have become a trendy topic in sports nutrition as even minor enhancement
of human physiology may positively affect high-intensity exercise and, therefore, competition results.
For example, there may be a decrease in the time of time-trial (TT) physical activities [27]. It is also
important to note that these changes seem to be highly related to the dosage and supplementation
protocol where exercise economy can be improved after a single dose of DN [28–33], but exercise
performance is more likely augmented after chronic use of DN [34–36]. Interestingly, it seems
ergogenicity is somehow related to the fibre-type ratio in muscles, augmenting the exercise economy
and performance more likely via type II muscle fibres (MFs) than type I [37].
During high-intensity exercise, oxidative phosphorylation is diminished as the O2 supply is
inadequate, and reliance on the anaerobic metabolic pathway of ATP regeneration is favoured [38].
Long-duration high-intensity exercise and intermittent high-intensity exercise lead to exercise-induced
hypoxemia causing the muscles to become hypoxic and acidic [39]. This impairment in homeostasis
may also disrupt the functioning of the NOS-dependent pathway, and continuation of exercise is
highly dependent on the activity of type II MFs [40,41]. Therefore, the reliance on the substitutional
NO3 − –NO2 − –NO pathway independent of O2 supply is increased [40,42]. Moreover, type II MFs have
a lower blood supply which affects the partial pressure of O2 within the microvasculature (Pmv O2 )
causing a lower O2 supply compared to type I MFs [43–45]. This phenomenon underlines the reliance on
the NO3 − –NO2 − –NO pathway in type II MFs and even more under hypoxia [24,46]. Lastly, most recent
studies suggest improvement in muscle force production and mitochondrial oxidative phosphorylation
are more likely observed in type II MFs than in type I MFs after DN supplementation [47,48].
Furthermore, it has already been suggested in earlier studies that neither acute nor chronic
DN supplementation can improve performance in highly trained cyclists [49–51], runners [52,53] or
cross-country skiers [54]. These groups of endurance-trained athletes tend to have a higher type I MF
ratio [55–57]. In contrast, high doses of DN (8.4–9.6 mmol) improved performance in highly trained
kayakers and rowers [29,58] but not low doses (~4–5 mmol) [29,59]. In this context, the upper body
muscles (e.g., biceps brachii, triceps brachii, deltoid, trapezius or latissimus dorsi) are well described
as muscles with a higher type II MF ratio [60–62]. Moreover, as highly trained athletes develop specific
adaptation to rowing [63] and kayaking [64], increased type II MF ratio or MF hypertrophy is more
likely [65]. This exercise modality, which mostly involves muscle groups with a higher type II MF
ratio [65], can be another example of the fibre-type specific effects of DN [37,66].
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1.3. Training Status as a Limiting Factor

Nutritional strategies to increase the bioavailability of NO and possibly physical performance
have been under the scope of research for many years, and there are some crucial variables
(e.g., muscle fibre-type ratio, physiological limitations) which can influence their effectiveness [8,67].
Consumption of dietary NO3 − (DN) in the form of either nitrate-rich foods or supplements generally
increases plasma levels of NO2 − [28] which interestingly does not always lead to improvement in
exercise performance especially in well-trained endurance and elite endurance athletes [68].
Fibre-type specific effects of DN supplementation have been demonstrated in animal experiments
where muscle force development increased in type II (fast-twitch glycolytic fibres type IIx) but not in
type I MFs (slow-twitch oxidative fibres) [34]. Reliance on the NO3 − –NO2 − –NO pathway is higher
in type II MFs due to the lower O2 tension (pO2 ) than in type I [43]. Therefore, an increase in the
bioavailability of NO mainly affects type II MFs [37]. Endurance-trained athletes are likely to have a
higher ratio of slow oxidative type I MFs compared to non-trained and recreationally active athletes [69]
or the inactive population and the elderly [70]. This phenomenon also relates to higher aerobic fitness
in highly and elite trained athletes which cannot be augmented any further [68]. These seem to be
possible explanations for the lower ergogenicity of DN supplementation in highly trained or elite
athletes, as some studies failed to enhance the performance of the participants [52,71]. It has been
suggested that the efficiency of DN supplementation is related to an athlete’s training status, especially
in high-intensity endurance exercises, e.g., time trial (TT) performance, where O2 delivery is impaired
and reliance on type II MFs is higher [68].
1.4. Aim and Purpose of the Review

This article reviews the relationship between VO2max and DN ergogenicity in TT performance,
defined as the athlete’s training status. We further discuss the relationship between simulated high
altitude and DN ergogenicity in the context of increased reliance on type II MFs under hypoxic
conditions, as it has been proposed that the DN ergogenicity is fibre-type related. Additionally,
we wanted to stress the importance of inclusion of certain methodological tools in the evaluation of the
results of experiments (e.g., smallest worthwhile change and typical error of measurement).

A review of the literature was carried out using articles indexed by Scopus and PubMed published
from 1 January 2015 to 31 December 2019. The PRISMA guidelines were followed to process the
review [72].
2.1. Search Strategy, Data Extraction and Analysis

The following terms were used for the search: dietary nitrates, physical performance and physical
exercise. All descriptors were searched using the Boolean operators to maximise the search quality
as follows: ((dietary AND nitrates) AND ((physical AND exercise) OR (physical AND performance))).
The title and abstract of each article were assessed for eligibility. After that, full-text articles were
reviewed to determine their suitability for inclusion or exclusion. The inclusion criteria were: (i) studies
on healthy human subjects, (ii) original, English-language research articles, (iii) inclusion of the
training status of participants (VO2max , training load or other affiliation to training status group),
(iv) experimental protocol consisting of time-trial testing, (v) results showing a change in time trial
performance, (iv) supplementation protocol is described. The exclusion criteria were: (i) the above
inclusion criteria were not met and (ii) the experimental test was combined with environmental
modification (e.g., high-altitude simulation and high temperature). No restrictions were made on age,
sample size, supplementation protocol or supplement form. Figure 1 describes the process of selecting
the articles presented in this review using the PRISMA Flow Diagram.
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Figure 1. Selection process diagram of articles meeting the eligibility criteria for our review.
2.2. Data Classification

Out of the 13 included studies, we analysed 22 separate DN experimental situations.
The experimental situations were classified into five groups according to the mean group VO2max or
VO2peak of the participants according to the preliminary testing of each study (Table 1), which allowed
us to define their training status as previously published [73,74]. One study did not use the VO2max of
participants for training status assessment but used training load instead [75]. Mean percentual change
in time-to-completion of given exercise tasks in the experimental DN groups were either included in
the article or calculated from the results.
Table 1. Participant training status classification defined by VO2max .
Training Status (Number of Experimental

Sex VO2max (mL/kg/min)
Situations Included)
1—Untrained (n = 1) Males <45
Females <37
2—Recreationally trained (n = 4) Males 45–54.9
Females 37–48
3—Trained (n = 4) Males 55–64.9
Females 48–52
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Table 1. Cont.
Training Status (Number of Experimental

Sex VO2max (mL/kg/min)
Situations Included)
4—Well-trained (n = 12) Males 65–71
Females 52–58
5—Elite (n = 1) Males >71
Females >58
2.3. Quality Assessment

Risk of bias was assessed using the Physiotherapy Evidence Database (PEDro) scale,
which provides a reliable assessment of internal validity [76,77]. Each article was individually
assessed by the reviewer (T.H.) using an adjusted 11 item checklist to yield a maximum score of 10.
2.4. Data Extraction

Data were extracted and reviewed by one of the researchers (T.H.). Additionally, the data were
independently reviewed by the other researchers (M.K. and P.V.), following a systematic selection list
which included the inclusion criteria mentioned in Section 2.1.

A one-way ANOVA was conducted to determine whether there is a relationship between DN
ergogenicity and training status level. Data were classified into three groups according to the training
status of participants: untrained + recreationally trained (n = 5), trained (n = 4), well-trained + elite
(n = 13). There were no outliers as assessed by a boxplot. Data were normally distributed for each
group as assessed by the Shapiro–Wilk test (p > 0.05), and variances were homogeneous as assessed by
Levene’s test of homogeneity of variances (α = 0.05). A Spearman’s rank-order correlation and Pearson
correlation coefficient were used to assess the relationship between DN ergogenicity and VO2max after
chronic/acute DN intake and between DN ergogenicity and TT test duration. A coefficient of variation
(CV) was carried out to assess the frequency distribution of VO2max among the groups of participants
and inter-study variability in performance effect. All data analyses were performed using the SPSS
statistical package (version 25; SPSS, Chicago, IL, USA) and the GraphPad Prism 8 (GraphPad Software,
San Diego, CA, USA). Results are presented as mean ± standard deviation.
3. Results
The reviewed articles included 22 experimental situations presented in Figure 2. The figure shows
performance changes in all acute (blue bars) and chronic (red bars) experimental situations reviewed.
The effect of DN supplementation is displayed as the percentual mean change in the experimental
group. Improvement in performance is interpreted as a negative value as the TT test was finished
faster compared to placebo.
The mean changes in performance across the selected experimental situations were −2.63%,
−2.83%, −0.49%, −0.27% and −0.31% in untrained (n = 1), recreationally trained (I = 4), trained (n = 4),
well-trained (n = 12) and elite (n = 1) groups, respectively.
One-way ANOVA presented a statistically significant difference in performance change among
groups with different training status after DN supplementation F (2, 19) = 11.787, p = 0.001 and ω2
= 0.533. The positive performance change increased from well-trained + elite (−0.272 ± 0.671%) to
trained (−0.490 ± 1.238%) and untrained + recreationally trained (−2.786 ± 1.629%) groups, in that
order. Tukey’s post-hoc analysis revealed that the increase from well-trained + elite to the untrained
+ recreationally trained group (2.51, 95% CI (1.12 to 3.91)) was statistically significant (p = 0.001),
as well as the increase from trained to untrained + recreationally trained (2.30, 95% CI (0.52 to 4.07),
p = 0.010), but not from well-trained + elite to trained. Statistical analysis was done on a sample of all
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22 experimental situations. Therefore, the data were divided according to the training status but not
according to the supplementation protocol due to the small sample size for ANOVA analysis.
Figure 2. Effects of DN supplementation on TT performance in athletes with different training statuses

according to their VO2max . The graph presents the mean percentual changes in time-to-completion
of given tasks in the experimental groups. Negative values represent improvement, as the task was
finished in less time (bars to the left from y-axis) following acute (blue bars) or chronic (red bars)
supplementation. Cited studies are listed in References [27,58,68,71,75,78–85]. * Statistically significant
improvement in performance following DN supplementation; # improvement was observed in 6 of
17 participants dividing the group of well-trained athletes into “responders” and “non-responders”.
The two experimental situations are, therefore, divided into four according to the specific reaction to
DN supplementation of the participants [81].
A Spearman’s rank-order correlation was run to assess the relationship between VO2max and
DN ergogenicity in the TT tests after chronic intake of DN. There was a statistically significant,
strong positive correlation between VO2max and performance change in TT, i.e., a positive ergogenic
effect declines with higher training status (rs (9) = 0.810, p = 0.003; Figure 3.). However, Pearson’s
correlation coefficient showed no significant correlation for acute supplementation (r(8) = 0.445,
p = 0.198; Figure 4). One study did not report VO2max values of participants and, therefore, was
excluded from the analysis [75].
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Figure 3. Relationship between VO2max and DN ergogenicity in TT tests: chronic supplementation

protocol. Negative values in time change represent improvement as the task was finished in less time.
Figure 4. Relationship between VO2max and DN ergogenicity in TT tests: acute supplementation

protocol. Negative values in time change represent improvement as the task was finished in less time.
Methodological Quality of Studies

All the reviewed studies that met the eligibility criteria scored 10 out of 10 in PEDro scale as they
followed double-blinded crossover placebo-controlled experimental design. Overall, the quality of the
reviewed studies was assessed as excellent.
4. Discussion
We reviewed the available research from 2015 to 2019 focused on the relationship between dietary
nitrates and TT performance in athletes with different training status according to their VO2max .
Statistical analysis of the data collected from 13 articles showed that the higher the training status,
the lower the effect on TT performance.
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4.1. Dietary Nitrates and Time Trial Performance

Our results demonstrate that DN ergogenicity in TT performance is more likely observed in
less-trained individuals than in the highly trained and more consistently after chronic use. These results
are in agreement with previously reported data [1]. In contrast, some reviews did not find a statistically
significant change in TT performance [86,87]. It was proposed that the VO2max > 65 mL/kg/min
is not related to ergogenic effects after DN intake [1,10]. However, our results suggest that DN
ergogenicity may be lower even in trained athletes, as we did not find a significant difference between
the trained and well-trained + elite groups. Significant ergogenic effects were evident in the groups of
VO2max ≤ 50 mL/kg/min and less.
These results suggest there is a strong link between the athlete’s training status and DN ergogenicity.
The real cause of this phenomenon is still debated. We further provide a discussion on possible causes.
4.2. Dietary Nitrates and Exercise Performance in a High-Altitude Environment

Our results are consistent with other studies suggesting the ergogenicity of DN is diminished in
highly-trained endurance athletes, possibly due to the fact of their higher type I MF ratio [1,37,88].
To support the hypothesis of a relationship between ergogenicity and type II MFs, we also
reviewed TT studies at simulated high-altitude through normobaric hypoxia (n = 3; Figure 5). A high
altitude environment with lower pO2 leads to impairment in O2 delivery in tissues, and the aerobic
energy metabolism pathway is diminished [89]. Therefore, the work efficiency of type I MFs is lowered,
and reliance on the anaerobic metabolic pathway and type II MFs is increased [90]. Exercise-induced
hypoxemia is amplified in high-altitude hypoxic conditions, and the reliance on type II MFs is expected
to be increased [91,92]. Therefore, it has been hypothesised that DN ergogenicity in highly-trained
endurance athletes could be augmented due to the increased involvement of type II MFs during TT
tests in an environment with lower p02 [93–95]. Although performance improvement was observed in
highly-trained athletes (VO2max > 65 mL/kg/min) after DN intake (−3.2%) [94], yet again, more consistent
improvement was more likely observed in less-trained athletes (−3.6 ± 0.4%) [93,94].
Figure 5. Training status and ergogenicity in simulated high-altitude. Cited studies are
listed in References [93–95]. * Statistically significant improvement in performance following
DN supplementation.
Additionally, Arnold et al. [95] observed an improvement in the highly trained (−0.4%), but this
was not of statistical significance (p = 0.6). To this day, data on the ergogenicity of DN at high altitude
brings more or less mixed results and, so far, no additional benefits in general [96,97]. Despite the
increased pulmonary NO availability and plasma NO2 − concentrations [98] and lower altitude-induced
reduction in endothelial function [99] that have been documented, solid conclusions as to whether a
high altitude could augment ergogenicity in highly-trained athletes requires further research.
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4.3. Dietary Nitrates Studies Methodology

Nyakayiru et al. [81] observed a statistically significant and positive response in six out of the total
of 17 participants during TT performance. Whereas the mean VO2max (65.0 ± 4 mL/kg/min) classified
the group as well-trained, the standard deviation suggested that some of the participants were less
trained and potentially more likely to be DN responders. In future research, we suggest the grouping
of participants to be carefully divided according to training status, because there seems to be a thin line
between the high and low ergogenicity of DN and, therefore, statistical evaluation of the results may
be affected.
Correctly classifying training status (e.g., VO2max and/or weekly training load) in various athletes
may be an essential aspect of increasing the reliability of DN research [73,74]. Table 2 summarises the
research characteristics of the reviewed studies, showing that specific TT criteria (distance, test duration)
and DN supplementation protocols (acute/chronic, DN dosage) are different across the studies. Notably,
groups of participants are somewhat mixed in terms of training status as the diversity of the athlete’s
VO2max is in some studies quite high (CV = 21.5% in Oskarsson and McGawley [82]).
Twenty-two experimental situations were included in our review with highly variable TT test
durations 881 ± 856 s (25–3300 s). No correlation was observed between the TT duration and
performance effects (rs (20) = −0.237, p = 0.288). There was also no correlation after the TT duration
adjustment (i.e., only situations with TT duration > 10 min included; rs (10) = 0.064, p = 0.843).
However, it may be speculated whether long-duration TT tests (>720 s) could benefit more from DN
supplementation as the authors of 10 out of the 12 experimental situations reported ergogenic effects.
In contrast, only 5 out of 10 experimental situations (TT test duration < 720 s) reported improvement
in exercise performance. It is becoming more evident that DN ergogenicity could be fibre-type specific.
Therefore, the duration of the event should be taken into consideration as our results are in contrast
with general recommendations for DN use (4–8 min events in Burke et al. [2]).
Inter-study variability in performance effect was high as the coefficient of variation (CV) reached
55%. The lowest and therefore more stable CV was obtained in trained (26%) and well-trained groups
(26%) but not in the recreationally trained groups (58%).
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Table 2. Relationship between DN supplementation protocol and training status in promoting the ergogenic effects.
Reference Sex Training Status VO2max a /VO2peak b CV A/C Supplementation Protocol Test (Test Duration) Ergogenic *
8.0 mmol (NO3 − ) of PN; 2 h
[75] M and F Recreationally trained - - 8 km cycling (~20 min) Yes
before the test
12.8 mmol (NO3 − ) of BJC; 3 h 1500 m running (~6 min) Yes
[84] M Trained 62.3 ± 8.1 mL/kg/min a 13.0%
before the test 10 km running (~45 min) No
M 59.0 ± 2.9 mL/kg/min a 4.9%
Nutrients 2020, 12, 2734
[82] 6.4 mmol (NO3 − ) of BJC; 2.5 h 1 km running (<3 min)

Trained No
F 53.1 ± 11.4 mL/kg/min a 21.5% before the test
Acute 9.9 mmol (NO3 − ) of PN; 2.5 h 180 m running (~25 s) No
[80] M Well-trained 69.3 ± 5.8 mL/kg/min a 8.4%
before the test 5 km running (~17 min) No
12.8 mmol (NO3 − ) of SN; 3 h
[81] M Well-trained 65.0 ± 4.0 mL/kg/min b 6.2% 10 km cycling (~17 min) No #
before the test
−)
6.4 mmol (NO3 of BJC; 2.5 h
[78] F Well-trained 52.3 ± 4.9 mL/kg/min a 9.4% 20 km cycling (~35 min) No
before the test
12.8 mmol NO3 − ) of BJC; 2 h
[58] F Well-trained † 47.8 ± 3.7 mL/kg/min b 7.7% 500 m paddling (~2 min) Yes
before the test
6-day 5.5 mmol (NO3 − ) of SN;
[68] M Untrained 37.8 ± 5.8 mL/kg/min b 15.3% 3 km running (~15 min) Yes
3.5 h before the test
15-day 8.0 mmol (NO3 − ) of PN;
[75] M and F Recreationally trained - - 8 km cycling (~20 min) Yes
2 h before the test
b 6-day 5.5 mmol (NO3 − ) of SN;

[68] M Recreationally trained 52.0 ± 4.5 mL/kg/min 8.7% 3 km running (~12 min) Yes
116
3-day 8.4 mmol (NO3 − ) of BJ; 2
[85] M Recreationally trained 45.4 ± 5.9 mL/kg/min a 13.0% 10 km running (~55 min) Yes
h before the test
8-day 6.4 mmol (NO3 − ) of BJC;
[83] M Trained 63.0 ± 4.0 mL/kg/min b 6.3% 4 km cycling (~6 min) No
Chronic 2.5 h before the test
7-day 12.8 mmol (NO3 − ) of BJC; 1 km cycling (~80 s) No
[71] M Well-trained 68.0 ± 3.0 mL/kg/min b 4.4%
2.5 h before the test 4 km cycling (<6 min) No
3-day ~5 mmol (NO3 − ) of BC; 1
[79] M Well-trained 65.2 ± 4.2 mL/kg/min a 6.4% 4 km cycling (<6 min) No
h before the test
6-day 12.8 mmol (NO3 − ) of SN;
[81] M Well-trained 65.0 ± 4.0 mL/kg/min b 6.2% 10 km cycling (~17 min) No #
3 h before the test
7-day 12.8 mmol (NO3 − ) of BJC;
[27] M Well-trained 66.4 ± 5.3 mL/kg/min a 8.0% 10 km cycling (~15 min) Yes
b 6-day 5.5 mmol (NO3 − ) of SN;

[68] M Elite 72.4 ± 6.1 mL/kg/min 8.4% 3 km running (~10 min) No
a VO2max values; b VO2peak values; * Statistically significant change in performance; # ergogenic effects in 6 of 17 participants following supplementation [81]; † 5 female athletes with
VO2peak 47.8 ± 3.7 mL/kg/min (recreationally trained or trained) are presented in the “well-trained” section of this figure because they were described as international-level female kayak
athletes and all were 2012 National Squad members with 3/5 athletes competing at the 2012 London Olympic Games [58]. VO2peak could have been lower due to the exercise modality [1].
VO2max /VO2peak values are presented as the mean ± SD. Abbreviations: dietary nitrates (DN); male (M); female (F); CV-coefficient of variation; beetroot juice concentrate (BJC); beetroot
juice (BJ); potassium nitrate (PN); beetroot crystals (BC); sodium nitrate (SN).
Nutrients 2020, 12, 2734
Dosage, time-to-test intake, supplementation protocol and form of DN also differed across the
reviewed studies. In eight studies, the dosage exceeded the amount of 8 mmol of NO3 − (up to
12.8 mmol; n = 5) which is the generally recommended amount whether an acute or chronic protocol is
chosen [6]. Time-to-test intake is 2–3 h in most studies (n = 11). In contrast, in some studies participants
were instructed to consume the supplement less close to the test (3.5 h in Porcelli et al. [68]) or closer
to the test (1 h in Callahan et al. [79]). A more consistent positive change in performance is likely
observed in athletes following a chronic supplementation protocol [1]. Our results showed a strong
positive correlation (rs (9) = 0.810, p = 0.003) between the VO2max and performance change after chronic
DN intake but no correlation in acute intake studies (r(8) = 0.445, p = 0.198). This suggests that chronic
DN intake provides more consistent results for all training status categories and underlines the lower
ergogenicity in highly trained athletes. The most common form of DN supplementation is beetroot
juice concentrate (BJC) [27,58,71,78,82–84]. Other forms of DN supplementation include raw beetroot
juice (BJ) [85], encapsulated potassium nitrate (PN) [75,80] and beetroot crystals (BC) [79] or a water
solution containing sodium nitrate (SN) [68,81]. All these variables in the supplementation protocol
could potentially interfere with the ergogenic effects. It has been well documented that individual
pharmacokinetic responsiveness after DN intake varies [10,28,31].
4.4. The Real Benefit for Elite Athletes?

Significant beneficial effects of DN intake in elite endurance athletes are less likely to be observed.
This athletic group demonstrated a high proportion of type I MFs, elite exercise performance close to
the athlete’s physiological limits or NO3 − -mediated vasodilation in non-prioritized muscles which
may lead to reduced O2 delivery to the essential muscles working very close to their maximal cardiac
output [100]. All these factors are now suggested as potential causes of the lower ergogenicity in highly
trained athletes.
Although the available laboratory studies do not support DN ergogenicity in well-trained athletes
by providing statistically significant outcomes, some might argue whether this is of concern in real
sport events. From the perspective of more ecologically valid situations (e.g., sports competitions),
even statistically insignificant improvement can change the overall result of TT performance. Therefore,
it may be speculated whether the 0.63% change in performance found in our analysis in the well-trained
group [27] could change the outcome in a real sports event. Other studies have discussed the same
phenomenon. For example, Rokkedal-Lausch et al. [27] concluded: “The improvement in 10-km TT
completion time and power output of 0.6% and 1.6%, respectively, in the present study, is of practical
relevance for elite and well-trained athletes”. Seemingly, the improvement in performance of only 0.6%
could make a significant difference when comparing the TT durations of elite cyclists. For example,
during the 17 km TT at the 2019 Giro d’Italia (Verona-Verona, winning time 22:07) or during the 31 km
TT at the 2019 Tour De France (Saint pée sur Nivelle–Espelette, winning time 40:52) only 0.3% and
0.04%, respectively, separated first and second position [101,102]. Nevertheless, in the laboratory
environment, the smallest worthwhile change (SWC) and the typical error of measurement (TEM)
should be taken into consideration when interpreting the results [103,104]. According to Hopkins [103],
the improvement in an athlete’s performance cannot be considered as progressive until it reaches or
goes over 0.3% in individual sports, defining this improvement as the SWC. Additionally, the TEMs for
TT cycling tests are usually around ~1% depending on the ergometer and time variation for short (~60 s)
and long-duration TT (~1 h) in elite athletes, also around ~0.5% and ~1%, respectively, depending on
the method and the equipment [105]. Furthermore, if we would focus on a longer duration TT test
(several minutes) then the athlete’s improvement would have to reach 1.3% (SWC + TEM), so it could
be called beneficial [103]. Therefore, even the most considerable TT performance changes observed
during cycling ergometry measurements in elite athletes may not be relevant (improvement is lower
than SWC + TEM, therefore unclear) in real sports events [103]. Interestingly only 5 of the 13 reviewed
studies observed performance improvement after DN supplementation ≥ 1.3% [68,75,81,84,85].
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From this point of view, it is evident that translating athlete-specific DN research outcomes into
practical interventions requires the determination of their true translational potential. To standardise
this, Close et al. [106] recently proposed an excellent 9 step framework, that may assist practitioners
in the proper evaluation of performance nutrition research and applying the findings into practice
therefore making the dietary choices adopted by their athletes sport-specific and “evidence-based”.
4.5. Future Perspectives

Recent results from muscle histology studies show fascinating new insights into the role of
NO3 − in the human body [107–111]. Seemingly NO3 − can be stored within the muscle tissue cells
which could be a very elegant explanation of the crucial role of muscle tissue in NOS production,
the NOS-dependent and NO3 − –NO2 − –NO pathways [88]. Wylie et al. [66] suggested that there is a
relationship between VO2max and NO3 − substrates within the MFs. In the light of these recent findings,
significant improvements in TT performance in athletes with VO2max < 65 mL/kg/min may be explained
by lower muscle NO3 − disposal and NO bioavailability. Therefore, increased DN intake may increase
muscle NO3 − storage capacity and positively affect physical performance [112]. Moreover, it has
been hypothesised there could be a potentially positive effect in the treatment of cardiovascular and
metabolic diseases as NO3 − levels are lower in the elderly, untrained and cardiovascular or diabetes
patients [111–113]. Therefore, further research in the area of NO3 − muscle storage and its role not only
in physical exercise but also in therapeutic practice is needed.
Based on the above discussion, it remains unclear whether any statistically insignificant changes in
highly trained athletes could be affected by MF ratio, non-specific tissue vasodilatation, physiological
systems already at their maximal limit of adaption in elite athletes, supplementation protocol, NO3 −
muscle storage or other factors. As such, future research should focus on unifying the study design
framework and strict classification of participants according to their training status. Additionally,
in the light of recent observations of the fibre-type specific effects of DN, it would be reasonable to focus
more on resistance and speed exercise tasks or lactate-anaerobic tasks to compare DN effectiveness
among different sports disciplines.
4.6. Limitations of the Study

Our study has limitations which should be noted. Firstly, we only focused on articles published
from 2015 and 2019, as we wanted to review the most recent studies and put them in the context of
future perspectives as the NO3 – muscle storage presents a new and fascinating approach in explaining
the DN ergogenicity. Secondly, our study did not focus on data related to NO3 − /NO2 − blood levels
which are the critical substrates for NO production and bioavailability. We managed to associate
performance enhancement with chronic use of DN. However, we may speculate whether increased
levels of NO3 − /NO2 − cause such a performance effect. We had not made the statistical analysis of
biochemical data to verify such association (some studies do not provide such data as the authors
did not focus on the biochemical analysis). Lastly, we analysed mean percentual changes in the
performance of the experimental groups gathered from the selected studies resulting in a certain bias
as the results can deviate across the group of participants.
5. Conclusions
In conclusion, reviewed studies from 2015 and 2019 show that ergogenicity of dietary nitrates in
time trial performance is more likely to be observed in lesser-trained athletes. Our results suggest that the
higher the athlete’s training status, the lower the exercise performance improvement. These results are
more consistent in chronic dietary nitrates supplementation studies rather than in studies following an
acute supplementation protocol. Furthermore, even a minor and statistically insignificant improvement
in performance of around 0.31% could make a difference for the elite trained, as the performance
differences between the podium athletes are tight. However, research results in elite athletes are
inconsistent (e.g., improvement/impairment, statistically significant/insignificant), and research samples
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are usually small. The smallest worthwhile change and typical error of measurement should be used
for critical assessment when evaluating time-trial research results. The performance change should
be considered as beneficial only when compared to the sum of the smallest worthwhile change and
typical error of measurement for selected training status groups and laboratory tests.
Author Contributions: Conceptualization, T.H. and M.K.; methodology, T.H., M.K. and P.V.; validation, P.V.;
formal analysis, T.H. and P.V.; writing—original draft preparation, T.H.; writing—review and editing, T.H., M.K.
and P.V.; supervision, M.K.; project administration, T.H.; funding acquisition, T.H. All authors have read and
agreed to the published version of the manuscript.
Funding: This article was written at Masaryk University as part of the project “Effect of Dietary Nitrates on
Physical Performance” MUNI/A/1201/2018 with the support of the Specific University Research Grant, as provided
by the Ministry of Education, Youth and Sports of the Czech Republic.
Acknowledgments: The authors would like to thank our colleague Alexander Floyd for reviewing the grammar
and critically reviewing the manuscript.
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nutrients
Article
The Influence of Cyclical Ketogenic Reduction Diet
vs. Nutritionally Balanced Reduction Diet on Body
Composition, Strength, and Endurance Performance
in Healthy Young Males: A Randomized
Controlled Trial
Pavel Kysel 1 , Denisa Haluzíková 1 , Radka Petráková Doležalová 1 , Ivana Laňková 2,3 ,
Zdeňka Lacinová 2,4 , Barbora Judita Kasperová 3 , Jaroslava Trnovská 2 , Viktorie Hrádková 3 ,
Miloš Mráz 3 , Zdeněk Vilikus 1, * and Martin Haluzík 3,4, *
1 Department of Sports Medicine, First Faculty of Medicine and General University Hospital,
12000 Prague, Czech Republic; [email protected] (P.K.); [email protected] (D.H.);
[email protected] (R.P.D.)
2 Centre for Experimental Medicine, Institute for Clinical and Experimental Medicine,
12000 Prague, Czech Republic; [email protected] (I.L.); [email protected] (Z.L.); [email protected] (J.T.)
3 Diabetes Centre, Institute for Clinical and Experimental Medicine, 12000 Prague, Czech Republic;
[email protected] (B.J.K.); [email protected] (V.H.); [email protected] (M.M.)
4 Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University
and General University Hospital, 12000 Prague, Czech Republic
* Correspondence: [email protected] (Z.V.); [email protected] (M.H.)
Abstract: (1) Background: The influence of ketogenic diet on physical fitness remains controversial.
We performed a randomized controlled trial to compare the effect of cyclical ketogenic reduction
diet (CKD) vs. nutritionally balanced reduction diet (RD) on body composition, muscle strength,
and endurance performance. (2) Methods: 25 healthy young males undergoing regular resistance
training combined with aerobic training were randomized to CKD (n = 13) or RD (n = 12).
Body composition, muscle strength and spiroergometric parameters were measured at baseline
and after eight weeks of intervention. (3) Results: Both CKD and RD decreased body weight, body fat,
and BMI. Lean body mass and body water decreased in CKD and did not significantly change in RD
group. Muscle strength parameters were not affected in CKD while in RD group lat pull-down and
leg press values increased. Similarly, endurance performance was not changed in CKD group while
in RD group peak workload and peak oxygen uptake increased. (4) Conclusions: Our data show that
in healthy young males undergoing resistance and aerobic training comparable weight reduction
were achieved by CKD and RD. In RD group; improved muscle strength and endurance performance
was noted relative to neutral effect of CKD that also slightly reduced lean body mass.
Keywords: body composition; ketogenic diet; strength parameters; endurance; training
1. Introduction
The last decade has been characterized by the search for alternative dietary ways to achieve
optimal body composition while maintaining or improving physical fitness and sports performance
to promote healthy lifestyle and prevent chronic diseases [1,2]. Current trends in sports nutrition
are increasingly reaching for the minimization of the carbohydrate component with ketogenic diet
becoming a very popular approach, in particular in endurance athletes [3,4].
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Nutrients 2020, 12, 2832
According to current definitions, carbohydrate intake within the range of 50–150 g per day can be
described as non-ketogenic low-carbohydrate regimens [5]. Ketogenic diet is most commonly defined
by a daily carbohydrate intake below 50 g per day or energy provision from carbohydrates for up to
10% of total energy intake [6]. Out of the frequently used approaches, targeted ketogenic diet allows
carbohydrates to be consumed immediately around exercise to sustain performance without affecting
ketosis [7]. The cyclical ketogenic diet (CKD) alternates periods of ketogenic dieting with periods
of high-carbohydrate consumption [8]. The period of high-carbohydrate eating is supposed to refill
muscle glycogen to sustain exercise performance [9].
The influence of ketogenic diets on sports performance is still the topic of an ongoing debate [10,11]
with often conflicting results [12]. The overreaching mainstream nutrition philosophy for endurance
athletes emphasizes a carbohydrate-dominant, low fat paradigm. Under these dietary conditions,
carbohydrates are utilized as predominant fuel source to cover high volumes of aerobic exercise [13].
The appeal of low carbohydrate high fat diet for endurance athletes is likely due to the shift in fuel
utilization, from a carbohydrate-centric model with limited glycogen sources to predominant fat
utilization with much bigger and longer-lasting fat stores [14]. This metabolic shift, seen after a period
of dietary alteration, is often referred to as being “fat-adapted”, which has been well-documented
in studies since the 1980s [15]. Substantial reduction in carbohydrate intake promotes utilization of
ketones and, according to some studies, it may enhance physical performance due to minimizing
the reliance of body metabolism on carbohydrates [16,17] and reduce lactate deposition leading to
enhanced recovery [18]. Importantly, ketogenic diets are, in particular in the short-term run, a very
efficacious way to reduce body weight not only in physically active subjects but also in patients with
obesity, type 2 diabetes and other chronic lifestyle diseases [19–21]. Nevertheless, it has to be noted
that long-term compliance and efficacy of ketogenic diet is not optimal and most of the studies had
rather limited duration [19,22].
Here we performed a randomized controlled trial to compare the effect of the cyclical ketogenic
reduction diet (CKD) vs. nutritionally balanced reduction diet (RD) on body composition, muscle
strength, and endurance performance in healthy young males undergoing regular resistance training
three times/week combined with aerobic training three times/week. We hypothesized that CKD will
be more efficacious in inducing fat loss as compared to RD while maintaining aerobic performance.
To this end, we explored the effect of eight weeks of CKD vs. RD combined with regular exercise on
body composition, and measures of strength and aerobic performance.

Twenty-five males of various fitness levels with minimum of one-year experience in resistance
training recruited from colleges of physical education and through a website with readers interested
in fitness and diets. Inclusion criteria were as follows: age between 18 and 30 years and a minimum
one-year experience with resistance and aerobic training. Subject recruitment began in April 2019 and
lasted until January 2020. Persons interested in participating were screened to ascertain they meet the
minimum criteria for the enrollment into the study.
Exclusion criteria were current injuries or health conditions that might have affected sports
performance or put them at risk for further injuries including the presence of cardiovascular diseases,
diabetes mellitus, arterial hypertension, or any other diseases that required pharmacological treatment.
Additionally, subjects taking any performance enhancing supplements (i.e., creatine, β-hydroxy
β-methyl butyrate, caffeine, protein powder, weight gainer, thermogenics, etc.), were required to
discontinue consumption at least one week prior to baseline testing and continue abstaining from
their use for the remainder of the study. The study was approved (ethic approval code 764/18 S-IV)by
the Human Ethics Review Board, First Faculty of Medicine and General University Hospital, Prague,
Czech Republic and was performed in agreement with the principles of the Declaration of Helsinki as
revised in 2008. Prior to randomization, all subjects were required to sign an informed consent.
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Nutrients 2020, 12, 2832
Using electronic randomization system, subjects were randomly assigned to follow either a CKD
or RD (both with total caloric intake reduction by 500 kcal/day) while participating in three strength
workouts and three aerobic workouts per week (30 min run, heart rate around 130–140 beats/min.)
for 8 weeks. Total caloric intake reduction by 500 kcal/day is counted from balanced hypocaloric diet
with a reduction of energy intake by 500 to 1000 kcal from the usual caloric intake. The U.S. Food
and Drug Administration (FDA) recommends such diets as the “standard treatment” for clinical trials
(FDA, 1996)
Subject randomization and follow-up during the study is depicted in CONSORT diagram in
Figure 1.
Figure 1. CONSORT diagram of subjects participating in an 8-week program while consuming a

cyclical ketogenic reduction diet (CKD) or nutritionally balanced reduction diet (RD).
2.1. Baseline and Postinterventional Testing

Data collection during baseline and post-intervention testing included medical history,
anthropometric examination, power performance test, bicycle spiroergometry, and blood drawings
to obtain laboratory data. BMI was calculated by the scale, using the height measurement. Accurate
height was measured using a basic stadiometer (Seca 222, Seca Co., London, UK).
2.1.1. Biochemical and Anthropometric Examination

At baseline, all subjects were weighted, and their BMI was calculated. Body composition was
measured using InBody Body Composition Analyzers (InBody230, InBody Co., Ltd., Seoul, Korea).
Body weight and other body composition measurements (lean body mass, body fat mass, BMI,
water content, and percentage of body fat) were taken with minimal clothes, no shoes, and measured
to the nearest 0.5 kg.
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Nutrients 2020, 12, 2832
Blood samples for biochemical measurements were taken prior to initiation of study and at the
end of the study after 8 weeks of diet. Serum was obtained by centrifugation and samples were
subsequently stored in aliquots at −80 ◦ C until further analysis. The maximal storage time was
8 months.
Biochemical parameters liver test, urea, creatinine, and circulating lipids were measured to exclude
liver, kidney, or lipid disorder. Creatine kinase and lactate dehydrogenase were measured to explore a
possible influence of the diets on muscle regeneration. β-hydroxy-butyrate was measured to confirm a
compliance to ketogenic diet.
β-hydroxy-butyrate was measured using TECOM Analytical Systems (TECOM Analytical
Systems CS spol. s r.o., Prague, Czech Republic). Other biochemical parameters were measured by
spectrophotometric methods using ARCHITECT c Systems device (Abbott Park, IL, USA.) in the
Department of Biochemistry of the Institute for Clinical and Experimental Medicine in Prague.
2.1.2. Strength and Aerobic Performance Testing

Power and performance testing were conducted over a 5-day period. Subjects signed for an
hour block to participate in each test. Each block had a maximum of 5 subjects in a gym and the
spiroergometry was reserved for each of them for an hour. Subjects were instructed to arrive at
the gym 30 min prior to testing times and not to train for at least 24 h before testing. A strength
performance testing for power output in the three exercises—bench-press, lat pull-down, and leg-press
was performed as follows: The subjects underwent an adequate warm up. After resting for two to four
minutes the subjects than performed a one-repetition maximum attempt of each exercise with proper
technique. If the lift/press was successful, after resting for another two to four minutes the load was
increased by 5–10% and another lift/press was attempted. If the subject failed to perform the lift/press,
after resting for two to four minutes they attempted the lift/press with weight reduced by 2.5–5%.
2.1.3. Methodology of Strength Testing

Upon arrival, the primary researcher explained the testing procedures and protocols and
demonstrated each test. Subjects were instructed to warm up. Power and aerobic performance
test administrators and personal researchers were blinded to the randomized group allocations.
Each proband participated in bench press, lat pull-down, and leg-press to assess the maximum
power performance.
2.1.4. Aerobic Performance Testing

Aerobic performance testing was carried out by bicycle spiroergometry using analyzer of
respiratory gases (Quark CPET, 1850 Bates Ave, Concord, CA 94520, Cosmed, USA). This metabolic
cart measures expired airflow by means of a pneumotach connected to the mouthpiece. A sample line
is connected to the pneumotach from which air is continuously pumped to O2 and CO2 gas analyzers.
Prior to testing, the pneumotach was calibrated with six samples from a 3 L calibration syringe. The gas
analyzers were also calibrated before each test to room air and calibration gases (15.21% O2 and 5.52%
CO2 , respectively). Heart rate (HR) was continuously recorded during exercise by electrocardiography
(Fukuda Denshi FX-8322 Cardimax ECG, 17725 N. E. 65th Street Bldg. C, Redmond, WA. 98052 USA).
Prior to exercise, the subjects were instructed to maintain a pedal cadence between 70 and 90 rpm
during exercise and to exercise to volitional fatigue. We used a modified exercise step protocol
0.33 W.min−1 as described by Gordon et al. [23]. The test was terminated when the subject was unable
to maintain a pedaling cadence of 40 rpm.
Maximal oxygen consumption was assessed by the attainment of the following criteria: (1) a
plateau (ΔVO2 ≤ 50 mL/min at VO2 peak and the closest neighboring data point) in VO2 with increases
in external work, (2) maximal respiratory exchange ratio (RER) ≥ 1.10, and (3) maximal HR within
10 b/min of the age-predicted maximum (220-age). All subjects met the first two criteria.
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Breath-by-breath gas exchange data from all tests were transferred to a spreadsheet program (MS
Excel 365) for further analysis. In addition, data from the VO2 max tests were time-averaged using 10 s
intervals to examine the incidence of an oxygen plateau.
2.2. Diet Protocol

Subjects were randomly assigned by electronic randomization system to either CKD or RD group
for 8 weeks. Subjects had a mandatory dietary session with a nutritionist prior to the beginning of
the study which provided detailed instructions on accurately keeping dietary food intake records.
All food record data were entered and analyzed using the DietSystem application (DietSystem App,
DietSystem App, s.r.o., Czech Republic).
2.2.1. Cyclical Ketogenic Reduction Diet

Total intake of energy was assigned to each participant based on lifestyle (individually calculated
according to somatotype, physical activity, type of work, etc.) and was reduced by 500 kcal per day.
Five days of low-carbohydrate phase, nutrient ratio (carbohydrates up to 30 g; proteins 1.6 g/kg;
fats: calculation of energy intake instead of carbohydrates) in order to induce and maintain ketosis.
Following with 2 days of carbohydrate phase (weekends): nutrient ratio (carbohydrates 8–10 g/1 kg of
non-fat tissue, 70% intake; proteins 15%; and fat 15%).
2.2.2. Reduction Diet

Principles of healthy nutrition, nutrient ratio (carbohydrates 55%, fat 30%, proteins 15% of total
energy intake). The overall caloric intake (individually calculated according to somatotype, physical
activity, type of work, etc.) was reduced by 500 kcal per day.
Both groups were given detailed instructions on acceptable foods for both types of diets.
In addition, subjects were given an 8-week low-carbohydrate meal plan or reduction diet meal
plan as per randomization.
2.3. Training Protocol
2.3.1. Development of Strength Skills

The plan was designed to develop maximum strength in the tested exercise and the muscles
involved. 3 differently focused trainings per week were performed:
• Focused on chest—bench press.

• Focused on the muscles of the lower limbs—leg press.
• Focused on the back muscles—lat pull-down.
One training unit lasted approximately 60 min. For each training unit, the full focus was on the
technique of execution and time under tension. Each training unit was performed with the maximum
possible effort to achieve the maximum results. The prescribed intensity in the form of load was
individualized and based on the entry measurements. The technical design, time under tension and
maximum effort must were similar for all subjects (maximum effort = maximum possible intensity
in compliance with technical parameters and number of repetitions) under tension and maximum
effort were similar for all subjects (maximum effort = maximum possible intensity in compliance with
technical parameters and number of repetitions)
2.3.2. Development of Endurance Skills

The plan consisted of a 30-min run at constant heart rate (at approximately 70% max TF or around
130–140 heart beats/minute).
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2.3.3. Supervision of Adherence to Training and Diet Protocols

Overall adherence to diet was checked once weekly by a nutritionist. Furthermore, adherence to
CKD was evaluated through urinary ketone measurements performed twice daily and by measurement
of blood β-hydroxybutyrate at the end of the study.
Training compliance was monitored through mandatory check-in procedures in a gym, and also
by a sport tester for aerobic performance (TomTom Runner Cardio, TomTom, The Netherlands).
2.4. Post-Intervention Testing

Data collection procedures were the same as baseline testing procedures. To ensure reliability,
power measures and performance testing were completed by the same researcher as at baseline for
each subject. In addition, subjects conducted their testing at the same time and with the same personal
researcher as pre-testing. Results from all tests were compared to the individual’s baseline values and
provided to the subjects after data analysis.

Statistical analysis was performed using Sigma Stat software (SPSS Inc., Chicago, IL, USA). Graphs
were drawn using SigmaPlot 13.0 software (SPSS Inc., Chicago, IL, USA). The results are expressed as
mean ± standard deviation (SD). Differences of body composition (body fat %, weight, BMI, lean body
mass, and fat mass), biochemical, and strength or aerobic performance parameters between CKD and
RD were evaluated using one-way ANOVA followed by Holm-Sidak test. Paired t-test was used for the
assessment of intra group differences as appropriate. Statistical significance was assigned to p < 0.05.
3. Results
3.1. The Influence of Cyclical Ketogenic Reduction Diet vs. Nutritionally Balanced Reduction Diet on
Anthropometric and Biochemical Parameters
Both CKD and RD decreased body weight (Figure 2), body fat mass and body mass index
with comparable effects of both approaches (Table 1). Lean body mass and body water content was
significantly reduced by CKD (Figures 3 and 4 and Table 1) while it was not influenced by RD.
Table 1. Anthropometric and biochemical parameters of subjects on cyclical ketogenic reduction diet
or nutritionally balanced reduction diet at baseline and after 8 weeks of diet.
Cyclical Ketogenic Diet

Reduction Diet (RD) ANOVA
(CKD)
V1-before V2-after V1-before V2-after
Number (n) 13 13 12 12
Age (year) 23 ± 5 NA 24 ± 4 NA NS
Height (cm) 181 ± 6 NA 186 ± 10 NA NS
BMI (kg/m2 ) 26.1 ± 3.7 24.6 ± 3.3 * 26.9 ± 4.3 25.5 ± 4.2 * NS
WEIGHT (kg) 85.6 ± 13.4 81.0 ± 12.0 * 93.0 ± 17.5 88.5 ± 17.4 * NS
MUSCLES (kg) 41.8 ± 4.5 40.0 ± 4.6 * 43.5 ± 5.3 43.1 ± 5.3 NS
FAT (kg) 12.9 ± 6.9 11.0 ± 5.8 * 17.6 ± 9.8 13.6 ± 9.0 * NS
% FAT 14.5 ± 5.5 13.0 ± 5.1 * 17.9 ± 6.9 14.2 ± 6.9 * NS
WATER (kg) 53.2 ± 5.6 51.0 ± 5.6 * 55.1 ± 6.4 54.8 ± 6.5 NS
CK (ukat/L) 4.40 ± 2.81 2.81 ± 1.21 3.80 ± 2.03 3.03 ± 2.03 NS
LDH (ukat/L) 2.68 ± 0.60 2.47 ± 0.42 2.74 ± 0.44 2.55 ± 0.33 NS
β-OH-butyrate (mmol/L) 0.2 ± 0.07 0.38 ± 0.25 * 0.24 ± 0.12 0.12 ± 0.04 NS
Data are mean ± SD. Statistical significance is from One-way ANOVA and paired t-test (V1—baseline testing vs.
V2—testing after 8 weeks of diet). * p < 0.05 vs. V1. BMI: Body mass index; CK: Creatine kinase; LDH: Lactate
dehydrogenase; β-OH-butyrate—β-hydroxy-butyrate. NS: Not significant. NA : not avalible.
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Figure 2. Individual responses of body weight for subjects before and after 8 weeks of cyclical ketogenic
reduction diet (CKD) and nutritionally balanced reduction diet (RD). Statistical significance is from
paired t-test * p < 0.05 vs. baseline.
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Figure 3. Individual responses of lean body mass for subjects before and after 8 weeks of cyclical
ketogenic reduction diet (CKD) and nutritionally balanced reduction diet (RD). Statistical significance
is from paired t-test * p < 0.05 vs. baseline.
131
Nutrients 2020, 12, 2832
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Figure 4. Individual responses of body water weight for subjects before and after 8 weeks of cyclical
ketogenic reduction diet (CKD) and nutritionally balanced reduction diet (RD). Statistical significance
is from paired t-test * p < 0.05 vs. baseline.
None of the diets significantly affected serum concentration of creatine kinase or lactate
dehydrogenase (Table 1), liver tests, urea, creatinine, or circulating lipids (data not shown).
β-hydroxy-butyrate significantly increased in CKD group while it was unaffected in subjects on
reduction diet (Table 1).
3.2. The Influence of Cyclical Ketogenic Reduction Diet vs. Nutritionally Balanced Reduction Diet on Muscle
Strength Parameters
The muscle strength parameters were assessed as maximum weight lifted during bench press,
lat pull-down, and leg press. CKD did not affect any of these parameters (Table 2). On the contrary,
in subjects on RD lat pull-down and leg press values significantly increased (Table 2).
Table 2. The effect of cyclical ketogenic reduction diet and nutritionally balanced reduction diet on
strength parameters.
Cyclical Ketogenic Diet (CKD) Reduction Diet (RD) ANOVA

Bench press (BP) 90.0 ± 24.2 90.0 ± 23.7 84.2 ± 21.8 87.7 ± 20.1 NS
Lat pull-down (LPD) 74.2 ± 15.7 76.0 ± 15.0 70.4 ± 14.8 75.2 ± 17.1 * NS
Leg press (LP) 138.0 ± 21.1 142.0 ± 16.3 127.8 ± 22.0 140 ± 22.8 * NS
V2—testing after 8 weeks of diet). * p < 0.05 vs. V1. NS: Not significant. NA : not avalible.
3.3. The Influence of Cyclical Ketogenic Reduction Diet vs. Nutritionally Balanced Reduction Diet on
Spiroergometric Parameters
Spiroergometric parameters are shown in Table 3. Respiratory exchange ratio decreased in subjects
on CKD while it did not change in subjects on RD. None of other spiroergometric parameters were
significantly affected in CKD group.
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Table 3. The effect of cyclical ketogenic reduction diet and nutritionally balanced reduction diet on
aerobic performance parameters.
Cyclical Ketogenic Diet (CKD) Reduction Diet (RD) ANOVA

TFmax 180.9 ± 10.2 178.0 ± 11.3 178.9 ± 11.8 179.0 ± 10.2 NS
Rmax 1.27 ± 0.08 1.2 ± 0.12 * 1.21 ± 0.04 1.16 ± 0.10 0.04
Wmax 297.0 ± 48.5 298.0 ± 54.3 282.1 ± 34.3 296.0 ± 35.9 * NS
VEmax 121.0 ± 28.5 136.0 ± 30.0 113.2 ± 20.3 124.0 ± 21.3 NS
VO2 max/kg 40.2 ± 4.1 43.0 ± 5.4 35.2 ± 6.0 38.2 ± 6.3 * 0.007
VO2 max/TF 19.0 ± 3.3 20.0 ± 3.4 18.0 ± 1.9 18.9 ± 1.6 NS
Wmax/kg 3.53 ± 0.42 3.6 ± 0.39 3.13 ± 0.52 3.36 ± 0.59 * NS
W170max/kg 3.27 ± 0.65 3.4 ± 0.37 2.8 ± 0.74 3.06 ± 0.83 * NS
V2—testing after 8 weeks of diet). * p < 0.05 vs. V1 TFmax—maximal heart rate; Rmax—respiratory exchange
ratio; Wmax—peak workload; VEmax—maximal pulmonary ventilation; VO2 max/kg—peak oxygen uptake;
VO2 max/TF—peak pulse oxygen; Wmax.kg peak workload/kg; W170 max/kg—physical working capacity (at a
heart rate of 170/min). NS: Not significant. NA : not avalible.
In contrast, in RD group peak workload, peak oxygen uptake/kg, peak workload/kg, and physical
working capacity at a heart rate of 170/min increased after 8 weeks of intervention.
4. Discussion
The most important finding of this study is that eight weeks of regular aerobic exercise combined
with exercise training complemented by two different dietary approaches—cyclical ketogenic reduction
diet or nutritionally balanced reduction diet—significantly decreased body weight and body fat in
healthy young men to a similar degree while having differential influence on body composition,
strength parameters, and aerobic performance.
Despite comparable influence of both diets on body weight, we detected distinctions in their effects
on body composition. In CKD group, the drop of body weight was due to a combination of decreased
body fat, body water, and a slight, but significant, decline in lean body mass. On the contrary, in RD
patients neither body water nor lean body mass were significantly affected and the weight reduction
was predominantly due to body fat loss. The influence of ketogenic diet combined with different forms
of exercise on body composition has been studied both in athletes and in patients with obesity and other
comorbidities on numerous occasions. In some of the trials, isocaloric [24] or hypocaloric ketogenic
diet [25] did not significantly change lean body mass while reducing body fat. On the contrary and
in agreement with our current data, Perissious and colleagues found a reduction in lean body mass
in patients with obesity undergoing exercise program while being on low carbohydrate diet [26].
Differential effect of ketogenic vs. nutritionally balanced diet under hyperenergetic conditions has
also been described in a study in healthy men undergoing an eight-week resistance training program.
Under these conditions, lean body mass increased only in control diet while it was unaffected in the
ketogenic diet group [27]. Finally, ad libitum low carbohydrate ketogenic diet reduced body mass
and lean body mass without compromising performance in powerlifting and Olympic weightlifting
athletes [28]. In our study, a slight decrease in lean body mass did not impair strength parameters as
compared to baseline values. Nevertheless, we have noted that in RD patients both lat pull-down and
leg-press significantly increased after eight weeks of intervention as compared to no change in subjects
on CKD.
While neutral effect of CKD on strength parameters in our study could have been expected
based on the previously published data [29,30], we hypothesized that ketogenic diet could be more
efficacious in improving endurance parameters as compared to nutritionally-balanced reduction diet
as suggested by some previous trials [31]. The increasing popularity of ketogenic diets in endurance
athletes is based on the hypothesis that predominant fat utilization over the use of carbohydrates may
improve energy availability during endurance exercise along with accelerated recovery [10]. Bailey
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and Hennesy recently reviewed available data on the influence of ketogenic diet on endurance in
athletes. They included seven studies into their analysis and concluded that limited and heterogenous
findings prohibit definitive conclusions [16]. In our study, we found decreased respiratory exchange
ratio in CKD groups after eight weeks of intervention as compared with no effect of RD suggesting
a shift towards lipid oxidation, which is in agreement with the mode of action of ketogenic diet
and previously published data [32]. However, none of the endurance parameters as measured by
spiroergometry have been affected in CKD group. On the contrary, in RD group peak oxygen uptake
and peak workload significantly increased after eight weeks of intervention. Our data suggesting
lack of improvement of endurance performance by ketogenic diet go in similar direction with results
published by Burke and colleagues in 2017 [33] and reproduced by the same group in 2020 [34] where
they found decreased endurance parameters in elite race walkers after ketogenic diet. By contrast,
in one of the early studies, low carbohydrate diet improved endurance times during moderate exercise
in moderately obese patients along with significant reductions in body weight and body fat mass [35].
Nevertheless, despite more pronounced fat loss the improvement on endurance performance with low
carbohydrate diet was comparable to that of high carbohydrate diet group.
When interpreted the results of our study within the context of currently published data it is
important to consider its strengths and limitations. The randomized design and the good compliance
of the subjects to dietary and treatment regimens can be consider strong points of our trial. On the
other hand, the limitations include relatively short duration, low number of subjects and inclusion of
only male participants.
Taken together our data are in general agreement with most of the previously published studies [36]
showing little or no benefit of ketogenic diet on endurance capacity. However, it should be noted that
contribution of fatty acids to metabolic response may differ with respect to duration and intensity of
exercise [37,38], exact type of training and numerous other characteristics. The utilization of fatty acids
increases with prolonged bouts of exercise of moderate intensity suggesting that ketogenic diet might
be useful especially with longer duration of aerobic exercise.
5. Conclusions
In summary, our data show that in healthy young males undergoing resistance and aerobic
training comparable weight reduction can be achieved with ketogenic and nutritionally balanced
reduction diet. In RD group, improved muscle strength and endurance performance was noted relative
to neutral effect of CKD on these parameters. Furthermore, CKD also slightly reduced lean body
mass. Our study thus demonstrates that the cyclical ketogenic reduction diet effectively reduces body
weight but is not an effective strategy to increase anaerobic or strength performance in healthy young
men. All in all, further randomized studies of longer duration are still needed to explore whether the
response to different diets is affected by long-term adaptation responses and whether it differs in males
and females or subjects with various types and levels of fitness.
Author Contributions: Conceptualization, P.K., D.H., R.P.D., M.H., and Z.V.; methodology, P.K., I.L., Z.L.,
B.J.K., J.T., V.H., M.M., D.H., Z.V., and R.P.D.; writing—original draft preparation, P.K., M.H., and Z.V.;
and writing—review and editing, all authors. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by Funded by CZ-RO (“Institute for Clinical and Experimental
Medicine—IKEM, IN 00023001”) and RVO VFN 64165 to M.H.
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35. Phinney, S.D.; Horton, E.S.; Sims, E.A.; Hanson, J.S.; Danforth, E., Jr.; LaGrange, B.M. Capacity for moderate
exercise in obese subjects after adaptation to a hypocaloric, ketogenic diet. J. Clin. Invest. 1980, 66, 1152–1161.
[CrossRef]
36. Harvey, K.L.; Holcomb, L.E.; Kolwicz, S.C., Jr. Ketogenic Diets and Exercise Performance. Nutrients 2019,
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37. Egan, B.; Zierath, J.R. Exercise metabolism and the molecular regulation of skeletal muscle adaptation.
Cell Metab. 2013, 17, 162–184. [CrossRef]
38. Evans, M.; Cogan, K.E.; Egan, B. Metabolism of ketone bodies during exercise and training: Physiological
basis for exogenous supplementation. J. Physiol. 2017, 595, 2857–2871. [CrossRef]
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nutrients
Review
Nutritional Ergogenic Aids in Racquet Sports:
A Systematic Review
Néstor Vicente-Salar 1,2, *,† , Guillermo Santos-Sánchez 3,†,‡ and Enrique Roche 1,2,4
1 Biochemistry and Cell Therapy Unit, Institute of Bioengineering, University Miguel Hernandez,
03201 Elche, Spain; [email protected]
2 Department of Applied Biology-Nutrition, Alicante Institute for Health and Biomedical
Research (ISABIAL-FISABIO Foundation), University Miguel Hernandez, 03201 Elche, Spain
3 Departamento de Tecnología de la Alimentación y Nutrición, Universidad Católica de Murcia,
30107 Murcia, Spain; [email protected]
4 CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III (ISCIII),
28029 Madrid, Spain
* Correspondence: [email protected]
† To be considered as equal first author.
‡ Present Address: Departamento de Bioquímica Médica y Biología Molecular e Inmunología,
Universidad de Sevilla, 41009 Seville, Spain.
Abstract: A nutritional ergogenic aid (NEA) can help athletes optimize performance, but an
evidence-based analysis is required in order to support training outcomes or competition performance
in specific events. Racquet sports players are regularly exposed to a high-intensity workload
throughout the tournament season. The activity during a match is characterized by variable durations
(2–4 h) of repeated high-intensity bouts interspersed with standardized rest periods. Medline/PubMed,
Scopus, and EBSCO were searched from their inception until February 2020 for randomized controlled
trials (RCTs). Two independent reviewers extracted data, after which they assessed the risk of bias
and the quality of trials. Out of 439 articles found, 21 met the predefined criteria: tennis (15 trials),
badminton (three trials), paddle (one trial), and squash (two trials). Among all the studied NEAs,
acute dosages of caffeine (3–6 mg/kg) 30–60 min before a match have been proven to improve specific
skills and accuracy but may not contribute to improve perceived exertion. Currently, creatine, sodium
bicarbonate, sodium citrate, beetroot juice, citrulline, and glycerol need more studies to strengthen
the evidence regarding improved performance in racquet sports.
Keywords: racquet sports; ergogenic aid; performance; sport supplement
1. Introduction
Racquet sports are included in the family of ball sports and more specifically, among those using
an implement. They are characterized by the use of a manual racquet to propel an implement (a ball,
shuttlecock, etc.) between two or four players with the objective of placing it in a position with no
return possibilities for the opponent. There are two different game formats: (a) passing the implement
over a net in a divided field (tennis, badminton, paddle and table tennis) or (b) hitting the implement
onto a wall in a shared field (squash and racquetball) [1].
Racquet sports are acyclic disciplines with very intense workload cycles, which are interrupted
by small pauses that allow for an incomplete recovery. Therefore, metabolic demands in racquet
sports alternate between both anaerobic and aerobic energy sources. Anaerobic energy comes from
intramuscular ATP and phosphocreatine (PC), as well as from anaerobic glycolysis, the three of which
are used during high intensity, short duration points, changes of direction, and hits. On the other hand,
137
Nutrients 2020, 12, 2842
the aerobic system is involved during long points of moderate intensity, playing a primary role in
delaying fatigue, and indirectly, favoring concentration, technical skills, and maintaining workload
during a match [2–5].
As a result of this fact, the average heart rate (HR) during a match reaches up to 60–80% of HR maximum
(HRmax), increasing to 90% of HRmax in high-intensity situations [6–8]. Nonetheless, HRmax does not
provide clear information regarding real energy demands or the metabolic pathways involved, since this
parameter is affected by dehydration, heat stress, age, and playing techniques [9]. Measuring blood
lactate concentration during a match could report more accurately the energetic pathways used by
racquet sports players. Ranges vary from 1.0–4.0 mmol/L to 8.0–12.0 mmol/L during prolonged
high-intensity matches [2,10–12], supporting the key role of glycolytic pathways during the match.
An ergogenic aid is any training method, mechanical device, nutritional or pharmacological
approach, or psychological technique that can improve exercise performance capacity and/or improve
training adaptations [13]. Therefore, a nutritional ergogenic aid (NEA) is defined as those nutritional
supplements taken orally containing a nutritional ingredient that intends to complement diet.
The objective of these supplements is to improve sports performance without exerting harmful
effects on the individual [14].
The consumption of NEAs has been increasing in recent years around the world, which has
led to a great variety of research with the aim of estimating their intake and use. In fact, sales of
dietary supplements grew 6.1% in 2017, achieving an income of 39.8 billion dollars in the US [15].
A meta-analysis published in 2015 concluded that elite athletes used many more dietary supplements
than non-elite athletes, and the prevalence of use was similar in men and women [16]. The NEAs
most frequently used by high-level tennis players tend to be creatine and caffeine [17] while among
international rank squash players, sodium bicarbonate is also frequently consumed in addition to
the two aforementioned NEAs [18]. Normally, NEA recommendations in high-level racquet sports
players are directed by personal trainers, coaches, or sports dietitian–nutritionists. However, proper
counseling based on current scientific evidence is required.
In this line, several organizations such as the Australian Institute of Sport (AIS) or the World
Anti-Doping Agency (WADA) propose classifications of sports supplements grouped into different
categories according to effectiveness, legality, and safety. Nevertheless, there are not policies regarding
the regulation of alleged benefits and safety claims [19,20]. Thus, athletes find themselves under the
influence of companies’ advertising, which claims improved performance and recovery through the
consumption of a wide range of products without scientific evidence regarding their effect, dosage,
or instructions for use.
The main aim of this systematic review was to evaluate the scientific evidence concerning NEAs
in the improvement of performance of racquet sports athletes specifically through published RCTs.

The conduct and reporting of the current systematic review conform to the Preferred Reporting
Items for Systematic Reviews and Meta-Analyses (PRISMA) [21]. Five racquet sports were analyzed
regarding the effectiveness of certain nutritional ergogenic aids: tennis, badminton, squash, table tennis,
and paddle.
2.1. Systematic Search

Relevant articles were identified by title and abstract in the electronic databases Medline, Scopus,
and EBSCO (since inception to 20 February 2020) using the search strategy in Table 1. The electronic
search was supplemented by a manual review of reference lists from relevant publications and reviews
to find additional publications on the subject.
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Nutrients 2020, 12, 2842
Table 1. Combined terms used in the search for studies in the database. 1 Mesh terms were used in the
search; 2 Term not included in the Mesh search; 3 nutritional ergogenic aid (NEAs) filed in the A group
of the Australian Institute of Sport (AIS).
Pubmed 1 Scopus and EBSCO

NEA Sport NEA 3 Sport
Dietary
Dietary supplements Racquet Sports Racquet Sports
supplements
Caffeine Tennis Ergogenic aid Tennis
Creatine Caffeine Badminton
Beta-alanine Creatine Table tennis
AND AND
Sodium Bicarbonate Beta-alanine Squash and sport
2 Sodium
Ergogenic aid Paddle
Bicarbonate
Nitrate
Beetroot juice
Glycerol
2.2. Data Extraction

Two reviewers (N.V.-S. and G.S.-S.) independently extracted the following data from each study
using a predefined Microsoft Excel data extraction form including the number of participants within
each group, participant characteristics, racquet sport discipline, and supplementation intervention
characteristics, end points, measurement methods, and results in order to produce an overview table
of all eligible studies.

Studies were eligible for inclusion if they met each of the following criteria: (a) not using any
doping substances established by the World Anti-Doping Agency (WADA), (b) using a randomized
controlled trial (RCT) design that included one group taking supplementation and 1+ groups receiving
a placebo or not taking supplementation, (c) not including any ergogenic aids classified within group A
by the Australian Sports Commission (AIS) because of their high evidence grade [22], (d) not presenting
supplementation as a source of nutrients, such as bars, gels, or drinks rich in carbohydrates and
electrolytes, and (e) not being gray literature (abstracts, conference proceedings, or editorials) or reviews.
2.4. Quality Assessment and Publication Bias

Characteristics of the retrieved RCTs were evaluated using the ‘risk-of-bias’ assessment tool following
the recommendations by the Cochrane Handbook for Systematic Reviews of Interventions [23,24].
This evaluation was carried out by two reviewers (N.V.S. and G.S.S.) working independently in order
to present bias comprehensively. The following criteria were analyzed: randomized treatment order
and carry-over effect (selection bias), blinding of participants and research staff to group allocation
(performance bias), blinding of outcome assessor (detection bias), incomplete outcome data (attrition
bias), selective reporting (reporting bias), and other bias (it was assessed if there was controlled diet,
exercise use of supplements or drugs, and sport stratification when a mixture of disciplines was
analyzed). Then, the retrieved RCTs were classified as being of “high”, “unclear”, or “low” risk of bias.
Effect size was calculated using Cohen´s d test.
3. Results
3.1. Included Studies

A total of 438 studies were screened by title and abstract, and 377 were assessed for eligibility
criteria (full-text screening). From the retrieved articles, twenty-one met all the inclusion criteria
and were included in the systematic review (Figure 1, Tables 2 and 3). Thirteen RTCs were found
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Nutrients 2020, 12, 2842
in the Medline database (eleven for tennis and two for badminton), seven were found in the Scopus
database (three for tennis, one for badminton, two for squash, and one for paddle) where one article
was not available despite requesting it from its main author; and none were retrieved from the EBSCO
database (because all those found there were repeated). Additionally, one article that was not found
through the initial search but was found in a review published in the Medline database was added
for full-text analysis. The PRISMA flowchart was applied to illustrate the step-by-step exclusion of
unrelated/duplicate retrieved records, leading to the final selection of twenty-one RCTs that met the
predefined inclusion criteria (Figure 1).
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chart [21] of the study selection process.
140
Table 2. Included studies on nutritional ergogenic aids in tennis. BCAAs: Branched-chain amino acids; FFA: Blood free fatty acids; Glu: Blood glucose; Gly: Blood
glycerol; HR: Heart rate; Lac: Blood lactate; LTPT: Leuven Tennis Performance Test; LTST: Loughborough Tennis Skill Test; NO; Nitric oxide; Pl: Placebo;
RSA: repeated-sprint ability shuttle test; STPT: Skill Tennis Performance Test; Trp/BCAAs: Blood tryptophan/branched-chain amino acids ratio; u-EPI: Urine
epinephrine; u-NE: Urine norepinephrine. ↑: Significant increase compared to placebo/control group; ↓: Significant decrease compared to placebo/control group;
↔: without changes compared to placebo/control group.
Blinded/Double
Nutrients 2020, 12, 2842
Study NEA Dosage/Time Participants Age (yrs) Level Duration Exercise Protocol Measurements Main Outcomes
Blinded
- Lac ↔ Lac
- Glu ↔ Glu
3 matches (2 of
- 0.2 - Gly ↔ Gly
75 min/match and
(women)–0.25
1 of 90 min/match - FFA ↔ FFA
(men) mg/kg/0 National
16 (8 men/ 25.4 ± 1.9/ with only rest
[25] Caffeine min before ranking DB 1 day - u-EPI ↑ u-EPI
8 women) 20.4 ± 2.8 between match 2
match and (Germany)
and 3 of 30 min) + - u-NE ↔ u-NE
every 15 min
Accuracy and - Sprints ↔ Sprints
during a match
sprint test
- Accuracy hit ↔ Accuracy hit
- Perceived exertion ↔ Perceptual training intensity
- 5 mg/kg/60 - Sprints ↔ Sprints
141
min before - Serve quality ↔ Serve quality
pre-test.
National LTPT + Sprint test - Backhand stroke quality ↑ Backhand stroke
- 0.75
[26] Caffeine 13 men 20.4 ± 0.9 ranking DB 1 day + Court session
mg/kg/Each 1 h - Volley errors and fatigue ↑ Volley errors and fatigue
(Belgium) (120 min) + LTPT
after start
- HR ↔ HR
pre-test and
during protocol - Perceived exertion ↔ Perceptual training intensity
- Lac ↔ Lac
- Glu ↔ Glu
- CK ↔ CK
- Prolactin ↔ Prolactin
- 3 mg/kg/30 National
1 match of - Fluid loss ↔ Fluid loss
[27] Caffeine min before 12 men 18.3 ± 3.0 ranking B 1 day
160 min/match - Serve and stroke velocity ↑ Serve velocity in 4th set
match (Australia)
- Serve kinematics ↔ Serve kinematics
- Perceptual skills ↔ Perceptual skills
- HR ↔ HR
Table 2. Cont.
Blinded/Double
Blinded
Intermittent - Successful shots ↑ Total shot successes
- 6 mg/kg/60 16 (8 men/8 National treadmill exercise
[28] Caffeine 20.7 ± 1.7 DB 1 day - HR ↔ HR
min before test women) ranking (45 min) + Tennis
skills test - Perceived exertion ↔ Perceptual training intensity
Nutrients 2020, 12, 2842
(USA)
University 3 days of sleep
- 80 mg/30 min 12 (6 men/6 players restriction follow
[29] Caffeine 18–22 DB 1 day - Accuracy serve ↔ Accuracy serve
before test women) (UK) a day of accuracy
serve test
- Handgrip force ↑ Handgrip force
- Serve velocity ↔ Serve velocity
Tennis specific - Running speed ↑ Only in high intensity
Elite-level
test + Simulated
- 3 mg/kg/60 14 (10 men/4 Junior - Number of sprints ↑ Number of sprints
[30] Caffeine 16.4 ± 1.2 DB 1 day Match of
min before test women) players
best-of-3-sets - Distance ↔ Distance
(Spain)
system
- HR ↔ HR
- Sweat rate ↑ Sweat rate
↑ Accuracy serve (depending
142
- Tennis serve trial - Accuracy serve of conditions of time and
National
- 6 mg/kg/60 10 (5 men/5 + Shuttle run distance
[31] Caffeine 19.9 ± 1.8 ranking DB 1 day
min before test women) sprint + Tennis
(USA) - Shuttle run time ↔ Shuttle run time
serve trial
- Likert scale ↔ Feelings
- Quality of 1st and 2nd
- 20 g/day ↔ Service quality
National service
(4 × 5g/day)/ LTPT + Shuttle
[32] Creatine 8 men 20.4 ± 0.9 ranking DB 5 days
During 5 days run sprint - Stroke quality ↔ Stroke quality
(Belgium)
before test - Sprint power ↔ Sprint power
- Lac ↔ Lac
- Service test + - Serving velocity ↔ Serving velocity
- 0.3 g/kg in
Ball machine
loading phase DB - Stroke velocity ↔ Stroke velocity
22.5 ± ground stroke
(6 days)
[33] Creatine 36 men 4.9–28.8 ± ITN 3 5 weeks drill + - Sprinting velocity ↔ Sprinting velocity
- 0.03 g/day in
4.8 Intermittent
maintenance - Strength ↔ Strength
sprint test +
phase (28 days)
Strength test - HR ↔ HR
Table 2. Cont.
Blinded/Double
Blinded
- Lac ↑ Lac
- pH ↔ pH
Keeps serve consistency while
Nutrients 2020, 12, 2842
- Serve consistency
Pl ↓
- 0.3 g/kg/70 min College
- LTST + Keeps stroke consistency while
Sodium before test Tennis - Stroke consistency
[34] 9 men 21.8 ± 2.4 DB 1 day Simulated match Pl ↓
Bicarbonate - 0.1 players
(50 min) + LTST
g/kg/During test (Taiwan) - Serve Accuracy ↔ Serve Accuracy
- Stroke Accuracy ↔ Stroke Accuracy
- HR ↔ HR
- Lac ↑ Lac
- pH ↑ pH
Junior STPT + RSA + - Stroke consistency ↑ Stroke consistency
Sodium - 0.5 g/kg/120 National Simulated match - Stroke accuracy ↔ Stroke accuracy
[35] 10 men 17.0 ± 1.0 DB 1 day
Citrate min before test Ranking (60 min) + STPT +
143
(Brazil) RSA - Number strokes ↔ Number strokes
- Time of sprints ↔ Time of sprints
- Serve velocity ↔ Serve velocity
- Serve velocity
test + Counter - Jump height ↔ Jump height
- 70 mL ATP and
Beetroot movement jump + - Handgrip force ↔ Handgrip force
(6.4 mmol of National 1 day
[36] juice 13 men 25.4 ± 5.1 DB Isometric
NO3− )/3 h ranking - Agility ↔ Agility
handgrip strength
before test (Spain)
+ Agility and - Sprint velocity ↔ Sprint velocity
sprint test
- Handgrip force ↑ Handgrip strength
- Isometric - Peak vertical power ↔ Jump power
Masters handgrip strength
- Anaerobic capacity ↔ Anaerobic capacity
Citrulline- - 8 g/60 min ranking in + Counter
[37] 17 women 51.0 ± 9.0 DB 1 day
malate before test USTA movement jump + - Relative peak power ↑ Relative peak power
(USA) Wingate cycling - Explosive power ↑ Explosive power
test
- Sustained power ↔ Sustained power
Table 2. Cont.
Blinded/Double
Blinded
- Lac ↔ Lac
- Gly ↔ Gly
- Glu ↔ Glu
Nutrients 2020, 12, 2842
- FFA ↔ FFA
- 0.17 g/kg Perceptual-motor
BCAAs performance test - NO ↑ NO
(Leu–Ile–Val = (LTST modified) + - Trp/BCAAs ↓ Trp/BCAAs
BCAAs + National
10:7:3) + 0.05 Simulated match
[38] Arginine + 9 men 25.6 ± 0.7 Ranking B 1 day - HR ↓ HR
g/kg Arginine + (120 min) +
Citrulline (Taiwan)
0.05 g/kg Perceptual-motor Prevents a high decrease in
Citrulline/80 performance test - Stroke Accuracy stroke accuracy compared
min before test (LTST modified) with Pl
Keeps stroke consistency
- Stroke consistency
while Pl ↓
Keeps stroke velocity while Pl
- Stroke velocity
↓
- Perceived exertion ↓Perceptual training intensity
144
- Change in body ↑ Body weight vs. Pl
Weight
↑ Plasma osmolality vs. Pl
- Plasma osmolality
(only pre- and post-exercise)
↑ Plasma volume vs. Pl (only
- Change in plasma
Tennis specific pre- and post-exercise)
- 1 g/kg/150 min Ranking
test + Simulated volume
before test 4–5 in
[39] Glycerol 11 men 27.0 ± 2.0 DB 1 day match (75 min) +
- 0.5 g/kg/15 min USTA - Electrolytes ↔ Electrolytes
Tennis specific
after test (USA)
test - Urine volume ↓ Urine volume
- Sprint velocity ↔ Sprint velocity
- Agility ↔ Agility
- Stroke accuracy ↔ Stroke accuracy
- Serve accuracy ↔ Serve accuracy
Table 3. Included studies on nutritional ergogenic aids in badminton, squash, and paddle. Glu: Blood glucose; HR: Heart rate; Lac: Blood lactate. ↑: Significant
increase compared to placebo/control group; ↓: Significant decrease compared to placebo/control group; ↔: without changes compared to placebo/control group.
Badminton
Blinded/Double Exercise
Study NEA Dosage/Time Participants Age (yrs) Level Duration Measurements Main Outcomes
Blinded Protocol
- Handgrip maximal
Nutrients 2020, 12, 2842
↔ Handgrip force
force
- Smash jump
- Squat jump ↔ Smash jump
Handgrip force ↑ Squat jump
- Countermovement
National + Jump tests + height/power
- 3 mg/kg/60 ranking 1 day
[40] Caffeine 16 men 25.4 ± 7.3 DB Agility Test +
min before test Jump (CJ) ↑ CJ height/power
(Spain) Simulated
match (45 min) - Agility ↔ Agility
- Number of impacts ↑ Number of impacts
- HR ↔ HR
↔ Perceptual
- Perceived exertion
training intensity
145
- Lac ↔ Lac
- Glu ↔ Glu
- 4 mg/kg/60 ↓ Errors in
Badminton - Errors in anticipation
min before anticipation
National specific test +
exercise - Accuracy serve ↔ Accuracy serve
ranking Fatigue protocol
[41] Caffeine - 4 mg/kg 12 men 28 ± 9 DB 1 day
(United (33 min) + - Reaction time ↓ Reaction time
/during 2nd
Kingdom) Badminton
Badminton - Time sprints ↓ Time sprints
specific test
specific test - HR ↔ HR
↓ Perceptual training
- Perceived exertion
intensity
- pH ↑ pH
Student Treadmill
Sodium - 300 mg/kg/90 1 day - Lac ↑ Lac
[42] 30 men 21 players ? testing to
bicarbonate min before test
(Indonesia) exhaustion - Time to exhaustion ↑ Time to Exhaustion
- pH ↓ pH
Student Treadmill
Sodium - 300 mg/kg/90 1 day - Lac ↑ Lac
[42] 30 men 21 players ? testing to
citrate min before test
(Indonesia) exhaustion - Time to exhaustion ↑ Time to Exhaustion
Table 3. Cont.
Squash
Blinded/Double Exercise
Study NEA Dosage/Time Participants Age (yrs) Level Duration Measurements Main Outcomes
Blinded Protocol
- 0.3 g/kg/day - Lac ↔Lact

(4 × 0.075 g National
Nutrients 2020, 12, 2842
9 (8 men/1 Court set sprint - Sprint time ↓ Sprint time

[43] Creatine /kg/day)/during 21.3 ± 0.3 ranking DB 5 days
woman) test - Likert scale ↔ Feelings
5 days (UK)
before test - HR ↔HR
- Peak Power ↑ Peak power
- 1000 mg
creatine + Cognitive tests - Fatigue ↓ Fatigue
1500 mg + Cycle - Reaction time ↔Reaction time
guarana + ergometer
National - Reaction time under ↓ Reaction time
Creatine + 133 mg sprint test +
[44] 8 18.2 ± 3.7 ranking ? 1 day pressure under time pressure
Guarana Caffeine/Half Cognitive tests
(France)
dosage at 30 + Submaximal - Visual response reaction ↓ Visual response
min and rest at test with time reaction time
0 min before cognitive test
test. - Ocular motility ↓ Ocular motility
response time response time
146
Paddle
- Isometric handgrip
↔ Handgrip strength
strength
Specific paddle
training (45 - Volley precision ↑ % Correct hits
6 mg/Kg /30 Amateur min) + - HR ↓ % Errors
[45] Caffeine 12 men 27.7 ± 3.7 B 1 day
min before test (Brazil) Handgrip
strength and - Perceived exertion ↔ HR
Volley test ↔ Perceptual
training intensity
Nutrients 2020, 12, 2842
3.2. Risk of Bias and Quality Assessment of Studies

The risk of bias of the included studies is illustrated in Table 4. The RCTs of Pluim; 2006 [33]
and Hartono; 2017 [42] were parallel group trials, so the criteria of random sequence generation
and allocation concealment were used instead of randomized treatment order and carry-over effect
respectively. Most of the trials assessed showed an unclear level in the criteria of selection bias, both
in the randomized treatment order and in the evaluation of the carry-over effect. Only the trials by
Vergauwen; 1998, [26] Lopez-Samanes; 2020 [36] and Abian; 2015 [40] suitably described the tools
used for randomization treatment, while trials by Wu; 2010 [34], Yang; 2017 [38], Abian; 2015 [40] and
Muller; 2019 [45] used tests to check if the washout time between conditions was suitable. Moreover,
most of the studies also showed a high risk of detection bias except for trials by Gallo-Salazar; 2015 [30]
and Abian; 2015 [40], which specifically indicated that blinding was kept until the statistical analysis
was performed. Five studies [27,38,42,44,45] were at a high risk of performance bias due to incomplete
blinding or a lack of blinding.
Table 4. Quality assessment of the included studies. Cross-over studies where A = Randomized
treatment order; B = Carry-over effect; C = Performance bias; D = Detection bias; E = Attrition bias;
F = Reporting bias; G = Other bias. * Parallel studies where A = Random sequence generation and
B = Allocation concealment.
Study A B C D E F G
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33] *
[36]
[37]
[39]
[34]
[35]
[38]
[40]
147
Nutrients 2020, 12, 2842
Table 4. Cont.
Study A B C D E F G
[41]
[42] *
[43]
[44]
[45]
3.3. Participants
Age in all the examined studies ranged from 16.4 to 51.0 years old, so that included from junior to
master players. Level ranged from university to professional level in both sexes, with a majority of
players being males (n = 266) as compared to females (n = 27). Tennis was the racquet sport about
which more studies on NEAs were checked (n = 15), followed by badminton (n = 3), squash (n = 2),
and paddle (n = 1), but no study was found for table tennis. In the case of NEAs, caffeine was the
most evaluated supplement (n = 10) followed by creatine monohydrate (n = 4), plasma buffers (n = 3),
nitric oxide (NO) precursors (n = 3), and hydration agents (n = 1).
3.4. Nutritional Ergogenic Aids and Intervention Characteristics in Tennis

Caffeine was the most tested NEA with seven studies (Table 2). All trials had a duration of 1 day
with variations in concentrations and timing. Most studies selected used a caffeine dosage of 3–6 mg/kg
30–60 min before the tests [26–28,30,31], with improvements in specific tennis skills such as accuracy
serve, backhand stroke, serve velocity in last sets, total number of successful shots, handgrip force,
and number of sprints compared with control groups. Other protocols with continued administration
during tests but a smaller dosage (0.2–0.25 mg/kg) [25] or the same quantity of caffeine given to each
player (80 mg) [29] only show an increase in urine epinephrine or no changes compared with control
groups respectively.
Regarding creatine monohydrate, only two studies evaluated its efficiency. Neither a high dosage
for five days (20 g/day) [32] nor a load period of six days (0.3 g/kg) followed by a maintenance period
(0.03 g/kg) until completing five weeks [33] offered advantages compared to control groups.
NEAs related to plasma buffer function were evaluated by two one-day duration studies. A load
of 0.3 g/kg sodium bicarbonate 70 min before test and continuous intake of 0.1 g/kg during test showed
maintenance of serve and stroke consistency (number of balls landed within the singles court on the
designated side) compared to the control group [34], while 0.5 g/kg sodium citrate 120 min before test
increased stroke consistency [35]. Both NEAs increased plasma lactate significantly, but only sodium
citrate was accompanied by an increase in blood pH.
Three studies about NO precursors were found. The intake of 70 mL beetroot juice 3 h before
the test did not show differences compared to control [36]. On the other hand, only citrulline malate
supplementation (80 g 60 min before the trial) [37] or together with arginine and BCAAs (0.05 g/kg
80 min before test) [38] showed improvements compared with control. Citrulline malate improved
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Nutrients 2020, 12, 2842
handgrip strength and relative peak power, and citrulline + arginine + BCAAs avoided the decrease of
stroke accuracy and kept stroke consistency and stroke velocity.
Lastly, regarding hydration agents, glycerol was the only NEA found in just one study [39].
The consumption of 1 g/kg glycerol 150 min before the trial and 0.5 g/kg 15 min after it increased body
weight, plasma osmolality, and plasma volume, and decreased urine volume.
3.5. Nutritional Ergogenic Aids and Intervention Characteristics in Badminton, Squash, and Paddle
Caffeine was tested in badminton male players 60 min before exercise protocol with a dosage
ranging from 3 to 4 mg/kg [40,41]. It showed improvements in jumps and the number of impacts
accompanied with a decrease in errors in anticipation, reaction time, and time of sprints (Table 3).
In addition, in paddle, caffeine showed ergogenic effects. The intake of 6 mg/kg caffeine 30 min
before the exercise protocol in twelve amateur male players increased the percentage of correct hits,
diminishing errors [45].
Different plasma buffers were evaluated by one study in badminton male players [42]. A load of
0.3 g/kg sodium bicarbonate or 0.3 g/kg sodium citrate 90 min before the test showed an increase in
time to exhaustion with both supplements (51.3 and 44.4% respectively). Both NEAs increased plasma
lactate, but only sodium bicarbonate showed an increase in plasma pH.
Finally, the effect of creatine was evaluated in squash players by two studies, one of them with a
load of 0.3 g/kg/day for 5 days before test [43], and the other with acute supplementation of a mixed
product composed by 1 g creatine + 1.5 g guarana + 133 mg caffeine [44]. The creatine loading protocol
showed a decrease in sprint time, while acute supplementation with guarana and caffeine increased
peak power and decreased fatigue, reaction time under pressure, and time visual response.
4. Discussion
4.1. Effects of Caffeine in Racquet Sports

Caffeine has shown to be an effective ergogenic aid for aerobic and anaerobic exercise with
improvements in performance and the perceptions of exertion and muscle pain with dosage ranging
from 2.35 to 5 mg/kg [46,47]. A similar dosage range was used in most of the racquet sports studies that
showed positive effects and a low risk of bias [26,30,40,41] with the exception of Hornery; 2007 [27] due
to its methodology of randomization and blinding. Even using higher doses (6 mg/kg), the positive
effects are verified [28,31,45] but with a moderate risk of bias due to aspects of randomization
or blinding.
In tennis, caffeine improved power skills such as backhand stroke, serve velocity, handgrip force,
and the number and velocity of sprints, as well as mental aspects such as the accuracy serve or total
number of successful shots. Lower dosages such as 0.2–0.25 mg/kg before and during a tennis match
or approximately 1 mg/kg 30 min before a serve test only increased epinephrine levels in urine but
they have not shown any performance improvements [25,29]. Moreover, both studies have a moderate
risk of bias since the control of randomization, the carry-over effect, the differences in caffeine dosage
between sexes, and the lack of certain control groups could be affecting the results. In another study [26],
it was shown that the use of a carbohydrate drink with or without caffeine showed improvements in
sprints and serve quality compared with the placebo group. As there were no differences between
both conditions, it is not possible to evaluate the real effect of caffeine in this study. Furthermore,
although an increase in sweat rate has been observed with low caffeine dosages (3 mg/kg) in junior
tennis players [30], several studies have disproved a dehydration risk [14].
On the other hand, handgrip force was not affected in badminton and paddle [40,45], but squat
and counter jump height or power were significantly better than in the placebo group in badminton [40],
offering a specific advantage in this discipline due to the net height in this sport.
In short resistance training, positive results have been observed regarding caffeine consumption in
the reduction of perceived exercise exertion [47]. However, no changes were observed in racquet sports
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(long duration intermittent sports) with the only exception of using two intakes of 4 mg/kg caffeine
before and after the first half of a badminton specific test and combining them with carbohydrates [41].
Despite these null effects, the number of total successful shots (with medium effect size (d = 0.57)) and
volley precision (high effect size (d = 0.86)) were improved in tennis and paddle respectively using a
high caffeine dosage (6 mg/kg) [28,45].
Therefore, the use of an acute ergogenic dosage of caffeine (3–6 mg/kg) 30–60 min before a
match is better than the intake of smaller concentrations, despite its continuous use during the
match. Due to the large seasons with accumulative long duration matches such as tennis, caffeine
consumption could be a useful aid for all competitive levels, since it may maintain physical and mental
conditions. More studies with a high caffeine dosage during long periods of intermittent exercise
and in combination with carbohydrates are needed in order to prove caffeine capacity to elicit high
accuracy and synergetic effects.
4.2. Effects of Creatine Monohydrate in Racquet Sports

Commonly, creatine monohydrate supplementation has been used as a strategy to increase muscle
mass and strength during training, but it has been also reported to improve power and anaerobic
capacity [48–50]. Thus, the use of creatine in intermittent sports such as racquet sports is of high
interest since about 75% of top 100 rank tennis players take it [17]. However, up to the present, there is
no evidence for recommendation.
Neither specific tennis skills, such serve or stroke, nor general physical aptitudes, such as sprints
or strength (typical short-duration high-intensity movements), were improved with different protocols
involving only a creatine load (20 g/day for 5 days) or load and maintenance (0.3 g/day for 6 days
and 0.03 g/kg for 28 days) [32,33]. Both creatine protocols were used in two studies that had a low to
moderate risk of bias.
On the contrary, in one study on squash, the intake of 0.3 g/kg creatine for 5 days was capable of
improving the sprint time in a specific test on court [43]. Due to the heterogeneity of sprint protocols,
it is not possible to reach a solid conclusion regarding its effect and the effect of moderate bias due
to lack of information about the randomization method, carry-over effect and missing information
about placebo composition. Moreover, the combination of 1 g of creatine + 1.5 g of guarana + 133 mg
of caffeine in an acute dosage improved several physical and alertness aptitudes in squash players,
but it has not been ruled out that the stimulant effect of guarana and caffeine were behind them [44].
Therefore, this fact—together with the lack of blinded groups—led to a high risk of bias.
Further studies should evaluate the sprint capacity or service and stroke skills with a high dosage
(16 g/day or 0.3 g/kg/day) for a longer period (at least 14 days), as has been shown in previous
works [49,50]. Further studies should also consider protocols that emulate long games. In addition,
despite not showing any clear improvements in specific tennis skills, creatine consumption during the
pre-season could be beneficial for the maintenance or increase of lean mass [50].
4.3. Effects of Buffering Supplements in Racquet Sports

High-intensity intermittent exercise tends to accumulate acid (H+ ) and carbon dioxide (CO2 ) in the
muscle and blood. Bicarbonate coming from CO2 acts as the primary mechanism to counteract plasma
acidification. The efficiency of acute sodium bicarbonate supplementation is influenced by exercise
duration. Specifically, extended duration (>4 min) sports have shown diverse results, with sodium
bicarbonate improving performance in running and cycling, but not in rowing, rugby, water polo,
or basketball [51].
An acute dosage (0.3 g/kg) and a continuous intake for a >4 min specific tennis test (0.1 g/kg)
did not improve accuracy or perceptual exercise exertion but kept serve (small effect size (d = 0.42))
and stroke consistency (small effect size (d = 0.09)), which decreased in placebo condition [34] with
a low risk of bias. On the other hand, in badminton players, only an acute dosage of 0.3 g/kg
increased time to exhaustion in a treadmill test, but not in a specific test in a high risk of bias
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study, since the randomized method, allocation concealment, blinding method and control of diet,
other supplementation consumption, and exercise load were poorly controlled [42]. Although the
blood lactate level was higher than in the placebo groups in both studies, this could be due to the
carboxylate co-transporter, which extracts lactate and H+ from working muscle cell to circulation
after an increase in extracellular pH [52] and an increase of glycolytic activity. Despite no changes
observed in extracellular pH between placebo and sodium bicarbonate before and after tests in tennis
players [34], changes between pre- and post-tests with supplementation may be enough to activate
lactate extrusion due to an enhance of glycolytic metabolism.
Other buffer supplements such as sodium citrate, used for causing less gastrointestinal distress
than other supplements, also showed significant high values of blood lactate compared to placebo
after an acute dosage (0.3–0.5 g/kg) 90–120 min before exercise [35,42]. Nevertheless, an increase in
extracellular pH was observed in tennis players, [35] which decreased in badminton players [42],
but there are contradictions about it, since the text of the study indicates otherwise. On the other hand,
sodium citrate was able to increase stroke consistency (high effect size (d = 1.41)) in junior tennis
players, just as sodium bicarbonate did, but it did not present effects in accuracy and perceptual exercise
exertion in protocols of >4 min duration [35]. With a non-specific badminton test, sodium citrate was
able to improve the time to exhaustion in a treadmill test [42]. Both studies have a moderate to high risk
of bias due mainly to the blinding methodology and the control of the intake of other supplementation.
More studies with a higher number of subjects would be needed with the aim of achieving strong
evidence about improvements in tennis skills as well as evidencing possible synergies between different
buffers (for example, beta-alanine) and other NEAs.
4.4. Effects of Nitric Oxide (NO) Precursors in Racquet Sports

It is well known that NO plays a relevant role as a second messenger. Its production is also
related to an increase in blood flow, which enhances nutrient and hormone delivery. NO also has a
favorable impact on resistance and endurance training adaptations [53,54]. Recent systematic reviews
and meta-analysis about NO synthase-independent pathway supplements showed that potassium
nitrate and sodium nitrate were less effective than beetroot juice on endurance exercise. The use of
beetroot juice supplementation containing 12–6 mmol nitrate displayed significant improvements in
time to exhaustion in a cycling race of 5–30 min duration but slightly non-significant improvements in
time trial or graded-exercise performance [55,56].
In intermittent sports such as tennis, beetroot juice containing 6.4 mmol nitrate did not show
any improvements in either explosive movements (serve velocity, jump, sprint, handgrip force)
or perceptual exertion in high-level tennis players [36] with a low risk of bias. These results are
similar to the ones found in recent studies in which short and high-intensity movements (such as
countermovement jump, isometric strength, or muscular movement concentric velocity) were evaluated
after the consumption of beetroot juice containing 6.4–17.7 mmol nitrate [57–59]. It seems that the
effect of beetroot juice could be beneficial in endurance performance due to nitrate conversion to NO,
affecting improvement in aerobic adenosine triphosphate (ATP) synthesis due to a reduction of VO2 .
In intermittent and short-term exercise, where the anaerobic alactic system is the main source of energy,
the effects are less clear. Only one-third of the studies evaluated in a recent systematic review of
intermittent exercise protocols [60] showed significant results in different variables of power compared
with the placebo group during repeated-sprint tests.
On the other hand, NO synthase-dependent pathway supplements, such as arginine or citrulline,
have shown different results. While arginine supplementation has demonstrated improvements in
both aerobic and anaerobic performance with acute (0.15 g/kg) or chronic (1.5–2.0 g/day for 4–7 weeks
or 10–12 g/day for 8 weeks) protocols [61], acute protocols of citrulline supplementation (3–6 g) showed
a small effect size (0.2) on high-intensity strength and power performance in resistance exercise [62].
In master female tennis players (51.0 ± 9.0 years), acute protocol with 8 g of citrulline improved
handgrip strength and power peak in a specific anaerobic test, but not the capacity of sustained
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power or jump power [37]. Due to the lack of a washing time between conditions and control of the
consumption of other stimulant substances, the risk of bias is moderate. Further studies are necessary
to analyze the role of citrulline supplementation in the performance of younger racquet sports players.
Yang et al. (2017) [38] showed improvements regarding the prevention of a decrease in stroke
accuracy and keeping stroke consistency and velocity (as opposed to a worsening in the placebo
group) using 0.05/kg citrulline +0.05 g/kg arginine +0.17 g/kg branched-chain amino acids (BCAAs).
The study presented a low risk of bias. Additionally, perceived exertion after the test decreased
significantly. These results appear to be due to a lower plasma tryptophan/BCAAs ratio than placebo,
since theoretically, BCAAs compete for the same tryptophan transporter across the blood–brain barrier,
avoiding serotonin formation and, consequently, central fatigue instauration [63]. It is common to use a
mixture of several NEAs in one product with the objective to obtain a synergic effect, but further studies
are necessary in order to verify the true effects of citrulline or arginine by themselves, without the
presence of the BCAAs being able to distort them.
4.5. Effects of Glycerol Supplementation in Racquet Sports

Finally, glycerol is a naturally occurring metabolite that acts as a plasma expander and could
help athletes prevent dehydration and improve thermoregulatory and cardiovascular changes [14].
Until 2018, the World Anti-Doping Agency (WADA) considered glycerol a banned substance, since
it was hypothesized that it may alter athlete biological passport [64]. In any case, the results of its
supplementation are mixed both in endurance and anaerobic disciplines [14]. In intermittent sports such
as tennis, 1.0 g/kg glycerol before followed by 0.5 g/kg after 75 min of simulated match, in environmental
conditions in the range of 29–38 ◦ C and 50–90% relative humidity (emulating conditions of important
tennis tournaments such as The Australian Open Grand Slam or Miami ATP Masters 1000), was not
capable of improving accuracy in serves or strokes, sprint velocity, or agility, in spite of its effect
increasing pre- and post-exercise plasma volume and osmolality [39]. This study has a moderate risk
of bias, since its randomized method, carry-over effect, blinding method and control of diet, and other
supplementation and drug consumption were poorly controlled. More research is needed to determine
glycerol’s supposed potential efficacy in racquet sports during more time-prolonged matches or during
several matches on the same day or on consecutive days in hot conditions.
5. Conclusions
Caffeine is the NEA showing clearer evidence of benefits for racquet sport players. Acute dosages
(3–6 mg/kg) 30–60 min before a match may improve specific skills and accuracy but may not contribute
to improve perceived exertion. Even though some evidence concludes that other NEAs, such as creatine,
sodium bicarbonate, sodium citrate, beetroot juice, citrulline and glycerol, could play an interesting
role in improving performance, more studies are needed to strengthen the evidence (Table 5).
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Table 5. NEA recommendations from current evidence. Green: High level of recommendation due to
the high number and quality of studies and the effects produced; Orange: Low level of recommendation
due to the low number and/or quality of studies and the effects produced; Red: Not recommended due
to the low number and quality of studies and contradictory or low effects.
NEA Effects Posology

- Improves specific racquet sports skills
- Improves sprints and jumps
Caffeine 3–6 mg/kg 30–60 min before competition
- Improves mental performance and
maybe accuracy
Creatine - May improve sprints 0.3 g/kg for 5 days
- May improve specific racquet
Sodium Bicarbonate sports skills 0.3 g /kg 70–90 min before competition
- May hold up time to exhaustion
- May improve specific racquet
0.3–0.5 g/kg 90–120 min before
Sodium Citrate sports skills
competition
- May hold up time to exhaustion
Beetroot juice - No effects 6.4 mmol 3 h before competition
- May improve handgrip strength
Citrulline-malate 8 g 60 min before competition
- May improve peak power
1 g/kg 150 min before competition and
Glycerol - No effects
0.5 g/kg 15 min after it.
Author Contributions: Conceptualization, N.V.-S. and G.S.-S.; methodology, N.V.-S. and G.S.-S.; protocol drafting,
N.V.-S. and G.S.-S.; risk of bias, N.V.-S. and G.S.-S.; quality assessment, N.V.-S. and G.S.-S.; data extraction, N.V.-S.
and G.S.-S.; literature search, N.V.-S. and G.S.-S.; search flowchart, N.V.-S.; writing—original draft preparation,
N.V.-S. and E.R.; writing—review and editing, N.V.-S., G.S.-S. and E.R. All authors have read and agreed to the
published version of the manuscript.
Funding: This study was supported by the official funding agency for biomedical research of the Spanish
government, Institute of Health Carlos III (ISCIII) through CIBEROBN CB12/03/30038), which is co-funded by the
European Regional Development Fund.
Acknowledgments: CIBEROBN is an initiative of Instituto de Salud Carlos III, Spain.
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nutrients
Article
Pre-Sleep Low Glycemic Index Modified Starch Does
Not Improve Next-Morning Fuel Selection or
Running Performance in Male and Female
Endurance Athletes
Monique D. Dudar 1 , Emilie D. Bode 1 , Karly R. Fishkin 1 , Rochelle A. Brown 1 ,
Madeleine M. Carre 1 , Noa R. Mills 1 , Michael J. Ormsbee 2,3 and Stephen J. Ives 1, *
1 Health and Human Physiological Sciences, Skidmore College, Saratoga Springs, NY 12866, USA;
[email protected] (M.D.D.); [email protected] (E.D.B.); kfi[email protected] (K.R.F.);
[email protected] (R.A.B.); [email protected] (M.M.C.); [email protected] (N.R.M.)
2 Department of Nutrition, Food, and Exercise Sciences, Institute of Sport Sciences and Medicine,
Florida State University, Tallahassee, FL 32306, USA; [email protected]
3 Department of Biokinetics, Exercise and Leisure Sciences, School of Health Sciences,
University of KwaZulu-Natal, Durban 4041, South Africa
Abstract: To determine the effects of pre-sleep supplementation with a novel low glycemic index
(LGI) carbohydrate (CHO) on next-morning substrate utilization, gastrointestinal distress (GID),
and endurance running performance (5-km time-trial, TT). Using a double-blind, randomized,
placebo (PLA) controlled, crossover design, trained participants (n = 14; 28 ± 9 years, 8/6 male/female,
55 ± 7 mL/kg/min) consumed a LGI, high glycemic index (HGI), or 0 kcal PLA supplement ≥ 2 h after
their last meal and <30 min prior to sleep. Upon arrival, resting energy expenditure (REE), substrate
utilization, blood glucose, satiety, and GID were assessed. An incremental exercise test (IET) was
performed at 55, 65, and 75% peak volume of oxygen consumption (VO2peak ) with GID, rating of
perceived exertion (RPE) and substrate utilization recorded each stage. Finally, participants completed
the 5-km TT. There were no differences in any baseline measure. During IET, CHO utilization tended to
be greater with LGI (PLA, 56 ± 11; HGI, 60 ± 14; LGI, 63 ± 14%, p = 0.16, η2 = 0.14). GID was unaffected
by supplementation at any point (p > 0.05). Performance was also unaffected by supplement (PLA,
21.6 ± 9.5; HGI, 23.0 ± 7.8; LGI, 24.1 ± 4.5 min, p = 0.94, η2 = 0.01). Pre-sleep CHO supplementation
did not affect next-morning resting metabolism, BG, GID, or 5-km TT performance. The trend towards
higher CHO utilization during IET after pre-sleep LGI, suggests that such supplementation increases
morning CHO availability.
Keywords: exercise; carbohydrates; time trial; substrate utilization; fat oxidation; fatigue;
gastrointestinal distress; satiety
1. Introduction
The importance of pre-exercise nutrition for exercise performance has been well documented [1–6].
However, given that many competitive endurance activities (training and/or competition) are scheduled
early in the morning, there exists a major limitation: inadequate time in the morning prior to the
event to properly fuel for sport. In addition, endurance athletes seldom consume much, if anything,
before training or competitions of 75–90 min in duration [7–11]. Unfortunately, this behavior may result
in sub-optimal physiological conditions such as carbohydrate depletion, dehydration, and fatigue [12],
which will adversely impact training quality and performance. This issue highlights the need to
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develop strategies to provide adequate nutrition from foods, beverages, and/or supplements that
athletes can consume pre-sleep without inducing gastrointestinal distress (GID) or disrupting normal
sleep patterns [6,8]. As an intervention, the incorporation of a pre-sleep meal may provide an added
“window of opportunity” for optimizing next-morning pre-race carbohydrate (CHO) availability and
exercise performance.
Glucose is the body’s preferred energy substrate during endurance exercise. Currently,
high glycemic index (HGI) CHOs are utilized by most athletes for pre- and/or intra-exercise nutrition due
to their rapid breakdown which drastically increases blood glucose availability. However, complications
such as GID, may arise with the consumption of HGI CHO sources prior to or during exercise due to
the gastrointestinal sensitivity to nutrient intake [13]. Consequently, this raises the question as to the
role of low glycemic index (LGI) CHO for exercise performance and nutrient timing. Contrary to a
HGI, a LGI slowly digests carbohydrates, thus providing more stable and long-lasting glucose release,
which may lower GID, both of which may better support endurance exercise performance [14].
Previous literature agrees that consuming LGI CHO prior to exercise results in enhanced
fat oxidation [15–18], and likely improved exercise performance [14,19]. However, not all studies
agree [20–23]. Stevenson and colleagues [22,23] investigated the effects of a low vs. high glycemic
index evening meal, approximately 16 h prior (at 19:00 p.m.), on next-morning metabolic responses at
rest and during exercise in males [23] and females [22]. Though in these studies [22,23] participants
were fed a standard HGI breakfast (at 08:00 a.m.) three hours prior to a 60-min run at 65% VO2max .
Although the breakfast elicited immediate post-prandial effects (lower glycemic and insulinemic
responses), there was no significant effect of the previous evening’s dinner glycemic index on substrate
utilization at rest or during running [22,23]. Though, given that meals were consumed 16 h prior to
testing, and participants were given a standardized HGI breakfast in the morning, it is not surprising
that any residual metabolic effects could not be detected. Given these limitations, it remains unknown
if pre-sleep CHO supplementation can optimize next-morning endurance athlete fuel selection and
performance, and whether low or high glycemic index would be preferential.
Though a LGI is touted as an efficacious source of CHO, a novel hydrothermally modified
LGI starch supplement was developed to manage glucose levels by providing a slow and steady
release of glucose to the body and brain for up to ten hours at a time [24]. In fact, data indicate a
lower peak and less rapid rate of decline in blood glucose than conventional cornstarch, which is
already considered a LGI CHO [25,26]. Currently, only a few studies have included the use of this
novel LGI CHO supplement [15,17,21,27] all of which included supplement ingestion either before,
during, or after exercise. No studies have investigated the effects of pre-sleep ingestion of a modified
CHO on next-morning exercise metabolism and performance. However, there is a plethora of data
investigating protein pre-sleep. The only study, to date, that investigated pre-sleep CHO-type beverage
was conducted by Ormsbee et al. (2016) [28]. The authors investigated the effect of pre-sleep chocolate
milk (HGI and protein) on endurance performance and found that chocolate milk resulted in increased
carbohydrate oxidation in the morning, but effects did not translate to 10-km running performance
improvements in females [28]. Given these data, we want to explore the effects of pre-sleep LGI CHO
since it has the potential to have a positive impact based on the slow/long-lasting release of glucose,
the lasting satiety, and if a HGI increases CHO oxidation, it is reasonable to suspect that LGI CHO
would have the opposite effect, as noted in acute day of studies.
Accordingly, the purpose of this study was to determine the effects of nighttime pre-sleep
supplementation with a novel LGI CHO on the next morning: (1) resting metabolism and GID;
(2) metabolic and GID responses to incremental exercise; and (3) 5-km time trial running performance
in trained male and female endurance athletes. It was hypothesized that the nighttime pre-sleep
consumption of LGI CHO would, in the next morning, enhance fat utilization during exercise, decrease
GID, and improve 5-km running time compared to a HGI CHO and a placebo (PLA) control.
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2.1. Subjects
Trained male (n = 8) and female (n = 6) endurance runners between the ages of 18 and 45 years
were recruited to participate in this study from local running clubs, triathlon teams, by word of
mouth, flyers, and through an email distribution around the Skidmore College campus. Participants
were included if they met the peak volume of oxygen consumption (VO2peak ) qualifications (women:
VO2peak ≥ 40 mL·kg−1 ·min−1 and men: VO2peak ≥ 45 mL·kg−1 ·min−1 ). Menstrual cycle status was
recorded for all female participants, though they were scheduled independently of the menstrual
cycle phase; notably, 3 out of 6 female participants did not have a menstrual cycle due to hormonal or
contraceptive therapy. Participants were excluded if they smoked, had uncontrolled thyroid conditions,
had been diagnosed with cardiac or metabolic disorders, regularly consumed anti-inflammatory drugs
or any dietary supplements intended to improve performance, or had musculoskeletal injury that
limited performance. All experimental procedures and risks of participation were explained verbally
and in writing prior to participants providing written informed consent. Approval for this study was
granted by the Human Subjects Institutional Review Board (IRB# 1901-786) of Skidmore College and is
in accordance with the most recent revisions of the Declaration of Helsinki.
2.2. General Procedures

This was a double-blinded randomized placebo-controlled study included four total trials:
one familiarization trial and three experimental trials. For the experimental trials, participants were
randomly assigned to consume either (1) LGI, (2) HGI, or (3) PLA at least 2 h after their last meal
and within 30 min prior to sleep on the evening before returning to the laboratory. Participants then
arrived to the lab in the morning after an overnight fast (~7–9 h after supplement consumption) for an
incremental exercise test (IET) and 5-km time trial (TT) (See Figure 1). Prior to the first experimental
trial, participants were required to complete a one-day dietary food and exercise log. Participants
were asked to replicate this diet and exercise, as closely as possible, prior to subsequent exercise trials.
Participants abstained from the use of non-steroidal anti-inflammatory drugs, caffeine, alcohol, and/or
vigorous activity at least 24 h prior to each experimental trial.
Figure 1. Experimental Overview; RMR = resting metabolic rate; IET: incremental exercise test.
2.3. Supplementation
Over the span of the study, all participants were randomized to the order in which they received
each of the following three supplements: (1) 532 mL of water mixed with 75 g of a HMS (LGI; Orange
Flavor, SuperStarch® , The UCAN Co., Woodbridge, CT, USA) (270 kcal; 0 g PRO; 66 g CHO; 0 g FAT),
(2) 532 mL of water mixed with 75 g of a HGI glucose-based supplement (Orange flavor, Gatorade® ,
PepsiCo, Inc., Purchase, NY, USA) (270 kcal; 0 g PRO; 67 g CHO; 0 g FAT), and (3) 532 mL of water mixed
with a color and flavor-matched, non-nutritive PLA (PLA; Orange CRUSH flavor packet and Benefiber),
with a volume of the powder visually similar to the other experimental conditions. Beverages were of
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similar taste, appearance, and consistency. The supplements were pre-packaged in inconspicuously
labeled/coded opaque containers by a researcher not otherwise involved in the study.
2.4. Familiarization Protocol

Participants filled out a physical activity readiness questionnaire (PAR-Q), ACSM health
preparticipation screening questionnaire, and a menstrual cycle history form (females only). Height
was measured using a stadiometer (Seca 213, portable stadiometer, Chino, CA, USA), while body
composition and weight were measured using air displacement plethysmography (BOD POD; COSMED,
Chicago, IL, USA) [29].
Peak volume of oxygen consumption (VO2peak ) testing was performed to assess baseline
cardiorespiratory fitness and inclusion in the study. Gas exchange and ventilatory parameters were
measured with a metabolic cart system (TrueOne 2400 Parvomedics, Salt Lake City, UT, USA) [30].
For each individual trial, the metabolic system was calibrated by a flow-calibration with a 3-L calibration
syringe and gas analyzer calibration with gas mixture of known concentrations of oxygen (O2 ) and
carbon dioxide (CO2 ) (16% O2 ; 4% CO2 ) according to manufacturer specifications, in addition to
environmental data for standardization purposes. Participants were fitted with a nose clip, two-way
non-rebreathe valve, and mouthpiece which was supported by a headpiece in order to collect expiratory
gases for analysis by the metabolic cart. The VO2peak protocol was performed on a treadmill (Woodway
PPS Med, Waukesha, WI) and the protocol required a self-selected constant pace that was “comfortable
but challenging.” Once the appropriate speed was determined, grade was increased at a rate of 2%
every two minutes until the participant reached volitional fatigue [28]. During the last 15 s of each
stage, HR was measured using a chest worn HR monitor (H7, Polar USA, Lake Success, NY, USA) and
RPE was measured on a 1–10 categorical ratio scale.
2.5. Experimental Protocol

A recovery period of >72 h was required after the familiarization trial and between each testing
day for all participants. On average, the time between trials was 192 ± 168 h. As sleep may have
influenced exercise performance we asked participants to self-report their sleep duration prior to each
visit. Participants then returned to the laboratory the following morning in a fasted, but well-hydrated,
state between 05:30 and 08:30 a.m. Upon arrival, participants were asked to provide a urine sample
to measure urine specific gravity using a hand-held refractometer to confirm hydration status [8].
Participants were provided with 250 mL of water to consume at their leisure before exercise and an
additional 250 mL of water if their urine specific gravity indicated dehydration (>1.020). Thereafter,
baseline measurements for height, weight, body composition, resting HR, satiety, GID, resting energy
expenditure (REE), and capillary blood glucose (BG) were collected. GID and satiety were measured
via a 100-mm visual analog scale (VAS) during baseline and one-minute post 5-km TT for each of
the three experimental trials [31–33]. Each VAS scale was marked with “0 mm” (no GID; extreme
hunger) and “100 mm” (extreme GID; extremely full) and participants were asked to draw a vertical
line indicating their perceived GID and satiety accordingly. Both VAS and categorical scale have been
documented in the literature as reliable perceptual measures of pain or discomfort [31–33].
REE was collected while participants rested quietly in a seated position with the headpiece and
mask on for 15 min in a climate-controlled room with the metabolic cart system described above.
Respiratory exchange ratio (RER) was recorded and relative substrate utilization (%FAT and %CHO)
was estimated [34]. The last ten minutes were used for data analysis. Resting HR was then measured
followed by blood sampling via finger stick. Capillary BG concentrations were measured using a
commercially available glucometer (OneTouch Ultra 2 LifeScan, Milpitas, CA, USA) [35].
2.6. Incremental Exercise Test and 5-km Time Trial

Fifteen minutes following completion of baseline measurements, participants completed a
three-minute warm up at a self-selected pace on the treadmill. Following the warm up, participants
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completed an incremental exercise test (IET) comprising of three stages of three minutes each at exercise
intensities of 55, 65, and 75% of VO2peak [34]. HR, RPE, and GID were recorded during the last 15 s
of each three minute each stage. GID and RPE were measured during exercise using a categorical
scale [31–33]. Upon completion of the IET, participants were given a five-minute active rest period
where they were instructed to walk on the treadmill at a comfortable pace to allow their HR to return
closer to baseline. Participants were allowed to use the restroom quickly as long as a researcher
monitored them for safety. Following the active rest period, participants completed a 5-km TT. The TT
was conducted rather than time-to-exhaustion to better mimic competition and pacing demands [36]
and due to greater reliability in the repeatability of the results [37]. Participants were instructed to
treat each TT as a competitive event and accordingly provide maximal effort. Participants could only
see their distance during the TT and the time and speed were blinded. Additionally, participants ran
both the IET and 5-km TT at 1% grade to best simulate the oxygen cost of outdoor running [38]. HR,
RPE, and GID measurements were taken every 1 km. BG and HR were measured immediately post
exercise and 10 min post exercise. HR, GID (VAS), and satiety (VAS) were also recorded one minute
post 5-km TT.

A sample size estimation was conducted (G*power, sample size estimator v.3.1.9.4; Kiel, Germany)
for F-test family in a repeated measures design, using the following parameters: average effect size of
night time supplementation of 0.39 on CHO oxidation [28], alpha level of 0.05, and minimal power of
0.8, which revealed a minimum sample size of 13 participants. Enrollment was targeted beyond this
minimum in the possible event of dropout. All statistical analyses were performed using commercially
available software (SPSS v.25, IBM, Armonk, NY, USA). A one-way repeated measures ANOVA was
used to determine if differences existed at baseline across conditions (PLA, HGI, LGI). A two-way
repeated measures ANOVA was used to analyze the potential impact of condition (PLA, HGI, LGI),
exercise intensity (55, 65, 75% VO2peak ), and their potential interaction on HR, RPE, GID, RER, % FAT
and % CHO utilization. Two-way repeated measures ANOVA models were used to compare condition
(PLA, HGI, LGI), distance (each km), and their potential interaction on RPE, HR, and GID during
the 5-km TT. Lastly, a two-way ANOVA was used to determine potential differences across condition
(PLA, HGI, LGI), time (baseline, one and ten minutes post 5-km TT), and their potential interaction on
BG. Tests of normality were performed and Greenhouse–Geisser corrections were utilized if sphericity
was violated. As men and women were recruited for this study, exploratory multi-variate ANOVA
measures were conducted including sex as an independent covariate in the model for the above
analyses. Significant main effects were followed up using Tukey’s Honestly Significant Difference,
and p values were complemented by effect size, which in this model we used partial eta squared (η2 ).
Alpha was set at 0.05. Data are presented as means ± standard deviation.
3. Results
3.1. Participants
Fourteen healthy endurance trained males (n = 8) and females (n = 6) completed all visits for this
study. An overview of subject characteristics is presented in Table 1. There were no differences in
self-reported sleep duration between visits (p = 0.56, η2 = 0.05, Table 1).
Table 1. Subject Characteristics.
Characteristic Combined Male Female

Sex (n, M/F) 14 8 6
Age (years) 28 ± 9 29 ± 9 27 ± 10
Height (cm) 169.3 ± 10.4 176.0 ± 7.1 160.4 ± 6.8 *
Weight (kg) 64.3 ± 9.8 70.1 ± 7.8 56.5 ± 6.2 *
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Table 1. Cont.
Characteristic Combined Male Female

Body fat (%) 18.9 ± 5.6 14.7 ± 3.5 23.9 ± 2.5 *
VO2peak (mL·kg−1 ·min−1 ) 55.4 ± 6.9 59.5 ± 5.5 49.9 ± 4.3 *
Note: Data expressed as means ± SD. VO2peak , peak oxygen uptake. * p < 0.05 male vs. female.
3.2. Effects of Supplement on Baseline Measures

Resting metabolic data are displayed in Table 2. There was no significant effect of supplement
on baseline REE (PLA, 1689 ± 278; HGI, 1701 ± 308; LGI, 1732 ± 287 kcal·day−1 , p = 0.72, η2 = 0.03;
Table 2). There was a significant interaction of supplement and sex for baseline RER, and thus relative
substrate utilization where males displayed a higher %FAT (PLA, 47.6 ± 5.4; HGI, 51.3 ± 11.5; LGI,
48.9 ± 11.8%, p = 0.02, η2 = 0.28) utilization compared to females (PLA, 46.9 ± 13.9; HGI, 28.3 ± 13.7;
LGI, 34.5 ± 22.2%, p = 0.02, η2 = 0.28) at rest for HGI and LGI. However, all other baseline measures
were unaffected by the supplement at baseline (all p > 0.05, Table 2).
Table 2. Baseline Measurements.
Supplement
Variable PLA HGI LGI p Value
Visual Analogue Scale (VAS) (mm)
Gastrointestinal Distress (GID) 22.4 ± 25.8 20.6 ± 22.5 26.2 ± 26.0 0.59
Satiety 55.3 ± 13.3 53.6 ± 23.0 50.3 ± 27.2 0.73
Substrate Oxidation
FAT (%) 47.3 ± 9.5 41.4 ± 16.8 42.7 ± 17.8 0.16
Carbohydrate (CHO) (%) 53.2 ± 9.6 59.1 ± 16.9 57.8 ± 18.0 0.16
VO2 (mL/kg/min) 3.9 ± 0.4 3.8 ± 0.4 3.8 ± 0.4 0.84
Resting Energy Expenditure (REE) (kcal·day−1 ) 1689 ± 278 1701 ± 308 1732 ± 287 0.72
HR (bpm) 57.7 ± 8.8 57.3 ± 10.6 59.9 ± 10.0 0.06
Blood Glucose (BG) (mg·dL−1 ) 97.7 ± 8.1 99.4 ± 8.8 98.4 ± 9.3 0.85
Urine Specific Gravity (USG) (a.u.) 1.02 ± 0.01 1.02 ± 0.01 1.02 ± 0.01 0.91
Sleep (h) 7.2 ± 0.8 7.2 ± 0.9 6.9 ± 1.2 0.68
Note: Data are means ± SD. PLA: placebo; HGI: high glycemic index; LGI: low glycemic index; VAS: visual analogue
scale; GID: gastrointestinal distress; REE: resting energy expenditure; BG: blood glucose; USG: urine specific gravity.
Data expressed as means ± SD.
3.3. Effects of Supplement on the Response to the Incremental Exercise Test (IET)
On average, during the IET, the LGI supplement tended to utilize less FAT (PLA, 44.1 ± 10.5; HGI,
39.7 ± 13.0; LGI, 37.5 ± 13.7%, p = 0.17, η2 = 0.14) and more CHO (PLA, 56.4 ± 10.6; HGI, 60.1 ± 14.3;
LGI, 63.1 ± 13.9%, p = 0.17, η2 = 0.14; Figure 2) than the other two supplements, though this did not
reach statistical significance. During the IET, there was no significant effect of supplement on VO2
(p = 0.23, η2 = 0.11, Figure 2C) or RER (p = 0.17, η2 = 0.14, Figure 2D). There was a tendency for
an interaction of supplement with intensity for VO2 where values tended to be lower with the PLA
during the lower intensity but equalized in the latter stages (p = 0.08, η2 = 0.18, Figure 2D). Expectedly,
all metabolic parameters were significantly affected by exercise intensity (all p < 0.001, all η2 > 0.90,
Figure 2A–D). The IET elicited an increase in GID (p = 0.04, η2 = 0.23, Figure 3) and RPE (p = 0.00,
η2 = 1.00, data not shown). Supplementation had no effect on GID (p = 0.28, η2 = 0.10, Figure 3) or RPE
(p = 0.55, η2 = 0.05, data not shown).
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Figure 2. Metabolic Response to incremental exercise test (IET) at 55, 65, and 75% of VO2peak between
placebo (PLA), high glycemic index (HGI), and low glycemic index (LGI) supplements (n = 14).
(A) Relative fat utilization (%FAT), (B) relative carbohydrate utilization (%CHO), (C) respiratory
exchange ratio, and (D) VO2 . Data expressed as means ± SD. * effect of intensity, p < 0.001.
Figure 3. Gastrointestinal distress (GID; categorical scale) during incremental exercise trial (IET) at 55,
65, and 75% of VO2peak (n = 14) between placebo (PLA), high glycemic index (HGI), and low glycemic
index (LGI) supplements. Data expressed as means ± SD. * effect of intensity, p = 0.04.
3.4. Effect of Supplement on 5-km TT

Supplement had no impact on HR during the 5-km TT (p = 0.89, η2 = 0.01, Figure 4A). Although
there was a significant effect of running distance during the 5-km TT on GID (categorical scale) (p = 0.00,
η2 = 0.58), there was no significant effect of supplement or an interaction of supplement by distance on
GID (categorical scale) during the 5-km TT (Figure 4B). RPE was not impacted by supplement (p = 0.35,
η2 = 0.01, Figure 4C). Running performance during the 5-km TT was unaffected by supplement (PLA,
21.6 ± 9.5; HGI, 23.0 ± 7.8; LGI, 24.1 ± 4.5 min, p = 0.94, η2 = 0.01, Figure 4D).
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Figure 4. (A) Heart rate (HR), (B) gastrointestinal distress (GID, CS), (C) rating of perceived exertion
(RPE) and (D) time (min) for 5-km time trial performance between placebo (PLA), high glycemic index
(HGI), and low glycemic index (LGI) supplements (n = 14). Data expressed as means ± SD. * significant
effect for distance p < 0.05.
3.5. Effect of Supplement on Perceptual Responses of GID and Satiety to Exercise

Supplement had no significant effect on satiety from pre- to post-experimental trial (p = 0.39,
η2 = 0.08; Figure 5A). There was no significant effect of supplement or time on pre- to post-experimental
trial GID (VAS) (Figure 5B, p = 0.56, η2 = 0.03).
$
Figure 5. Cont.
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%
Figure 5. (A) Satiety (mm; VAS) and (B) gastrointestinal distress (GID; mm; VAS) at baseline (pre) and
at conclusion (post) of running the 5-km time trial performance between placebo (PLA), high glycemic
index (HGI), and low glycemic index (LGI) supplements (n = 14). Data expressed as means ± SD.
3.6. Blood Glucose (BG)

There were significant main effects for time (p = 0.00, η2 = 0.66), where blood glucose at baseline
(PLA, 97.7 ± 8.1; HGI, 99.4 ± 8.8; LGI, 98.4 ± 9.3 mg·dL−1 ) was significantly increased immediately
post-exercise (PLA, 127.2 ± 19.4; HGI, 131.0 ± 28.8; LGI, 124.4 ± 27.9 mg·dL−1 , p = 0.00) and ten minutes
post-exercise (PLA, 127.6 ± 23.9; HGI, 133.3 ± 24.6; LGI, 126.7 ± 23.3 mg·dL−1 , p = 0.00; Figure 6), but no
differences were observed between immediate and ten minutes post exercise. There were no significant
differences in BG between supplements (p = 0.54, η2 = 0.04) at any time point or an interaction (p = 0.87,
η2 = 0.02).
Figure 6. Blood glucose levels (mg·dL−1 ) at baseline, 1 min post 5-km TT run, and 10 min post 5-km TT
run trial between placebo (PLA), high glycemic index (HGI), and low glycemic index (LGI) supplements
(n = 14). Data expressed as means ± SD. * significant effect of time, p = 0.00.
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4. Discussion
The present study is the first to assess the effects of pre-sleep supplementation with a novel LGI
CHO as compared to HGI CHO or placebo control on next-morning (~8 h later) exercise metabolism,
GID, and endurance performance in male and female endurance athletes. It was hypothesized that
the nighttime pre-sleep consumption of LGI CHOs would increase fat utilization during morning
exercise, decrease GID, and improve 5-km TT performance. The primary findings were as follows:
(1) supplementation had no significant effect on REE, CHO, or FAT utilization at rest, though females
tended to utilize more CHO in the HGI and LGI supplement at rest; (2) supplementation had no
significant effect on substrate utilization during graded submaximal exercise; (3) blood glucose was not
different among supplements at any point during the trial; (4) perceptions of GID were not different
among supplements; (5) supplementation had no discernable significant effect on 5-km TT performance.
Although our data do not support our original hypothesis, the present study suggests that there are no
detrimental effects of supplementing with either LGI or HGI CHO pre-sleep in endurance athletes
and thus, they may be utilized as a feeding window and fueling strategy to ingest adequate daily
energy intake.
The gastrointestinal tract can be very sensitive to the foods and beverages we consume.
Unfortunately, nutrient ingestion prior to and during exercise may lead to GID. Baur and colleagues
(2016) reported that GID increased after the consumption of the same hydrothermally modified starch
(HMS) LGI supplement that our current study used [15]. Baur et al. (2016) compared the HMS to an
HGI CHO supplement when ingested prior to, and during, prolonged cycling in ten trained male
cyclists and triathletes [15]. It was reported that there were likely large correlations between mean
sprint nausea (r = −0.51) and total GID (r = −0.53) and exercise trial, showing that GID contributed to
reduced cycling performance [15]. Further, there was a HMS-associated increase in GID negatively
effecting sprint cycling performance [15]. Given that HMS is slow releasing under normal digestion
supplements, malabsorption may be the explanation for the primary pathophysiologic mechanism of
LGI CHO-induced GID during exercise. Unlike the findings of Baur et al. (2016), the present study
found no effect (positive or negative) on GID and performance. Perhaps the pre-sleep ingestion of LGI
CHO avoids the LGI CHO-induced increase in GID in morning endurance performance. This is likely
because the body can digest the LGI CHO during the overnight period. Participants in the Baur et al.
(2016) study consumed LGI CHO during the exercise as well, which likely caused the incidences of
GID with HMS ingestion [15].
An LGI CHO may still be an optimal source of CHO for athletes given its previously reported low
osmolality, low insulin impact, slow release factor, and maintenance of blood glucose levels [5,24–26].
In general, elevated insulin levels attenuate lipolysis and fat metabolism, thus increasing utilization
of CHO. Even though it is well documented that consuming LGI carbohydrates before exercising
results in enhanced fat oxidation, or at least maintaining euglycemia during exercise [17,18,21,39–43],
and possibly improved performance [44], though not all agree [16,20]. Data from the present study,
albeit in a different methodological approach, do not support these findings, as we found no effect of
LGI CHO, or HGI CHO for that matter. When comparing LGI to HGI, some studies have reported
enhanced exercise performance [19,39,43,45,46] while other studies report no differences [41,42,47–50].
For example, Baur et al. (2016) reported an increase in total FAT oxidation and reduction in CHO
oxidation with LGI supplementation 30 min before as well as during exercise [15], which disagrees
with the findings of the present study utilizing pre-sleep supplementation of LGI. These inconsistencies
may be explained by, principally, time but other methodological differences, such as timing or dose of
CHO supplementation, type of exercise protocol (i.e., cycling versus running), or sample size should
also be considered. Researchers have reported muscle glycogen sparing with LGI compared to HGI
CHO [47], which may be explained by improved fat oxidation. Our findings are in accordance with
previous literature that LGI and HGI CHO do not improve running TT performances [21,28].
Glycemic control is extremely important for those training and competing in endurance
competitions and increasing fat oxidation could potentially benefit performance by preserving
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glycogen stores [51]. To maximize glucose fueling, the timing of pre-exercise consumption of CHO is
essential, along with the type/amount of exercise being performed. The time of consumption may alter
the metabolic effects. Studies have shown that CHO consumed one to four hours prior to exercise
resulted in a decline in glucose and insulin basal levels prior to exercise [2,52]. Further research has
reported that CHO consumed ≤ 60 min before exercise leads to elevated blood glucose and insulin
levels immediately prior to exercise [47,53–55]. These findings emphasize the importance of nutrient
timing and the exploring how the body performs from nutrient consumption solely the night before
exercise takes place.
The trend towards higher CHO utilization during exercise after pre-sleep consumption of HGI or
LGI CHO, perhaps more so in LGI, might suggest that pre-sleep LGI CHO supplementation increases
morning CHO availability or more stable bioavailability, though more research is needed as this was
not directly investigated in the present study. Due to the exercise paradigm used in the current study,
the 5-km TT run lasting ~20–30 min could present itself as a higher intensity glycolytic exercise than
longer endurance exercise performance trials. Research on the effects of CHO feeding for endurance
exercise indicates that some measures of performance are more sensitive than others, and short duration
exercises may not be long enough to cause CHO depletion and reveal potential effects of pre-sleep
CHO supplementation [12]. This might explain the insignificant differences in 5-km TT performance
in the current study, and perhaps longer bouts, and/or larger sample sizes, are required to reveal an
effect. There was, however, a significant effect between supplement and sex for resting CHO and FAT
oxidation in this study, where females utilized more CHO with LGI and HGI (PLA was consistent
between sexes). This suggests females resting fuel selection may respond differently to pre-sleep LGI
or HGI CHO supplementation, but further work is needed.
In the present study, which utilized a graded and shorter duration endurance event, we found
no benefit with pre-sleep ingestion on enhancing exercise performance. A contributing factor for the
lack of significant positive impact on exercise performance may be attributed to the relatively short
duration of the exercise stimulus incorporated in the present study [12], the amount of CHO, and/or
sample size. When exercise is prolonged in a moderately intense state, CHO oxidation gradually
decreases while fat oxidation increases [51,56]. Muscle glycogen utilization decreases due to reduced
muscle glycogen availability [57] hence why CHO supplementation is vital for exercise of longer
duration since the body relies on CHO as fuel [13]. The exercise module that was used in the present
study was based on previous literature that found an effect of nighttime feeding altering morning
metabolism in a 10-km run [57], and was preceded by an incremental exercise trial of three five-minute
stages at 55, 65, and 75% VO2peak [57]. That protocol was altered to test a 5-km timed trial with an IET
comprised of three three-minute stages at the same intensities. A main reason why those times were
chosen include efficiency and time restraints. Additionally, not measuring substrate utilization during
the 5-km TT limited the current study’s understanding of substrate metabolism to only the initial
nine-minute incremental test but this was intentional to allow the athletes to give their best efforts and
be minimally distracted. Contrary to our hypothesis, we found that pre-sleep supplementation with
LGI CHO tended to the lowest FAT oxidation as compared to HGI and placebo control. In the present
study, we cannot ascertain the mechanisms responsible such as altered intramuscular CHO availability,
or altered bloods level of glucoregulatory hormones (i.e., insulin and glucagon).
Experimental Considerations
Future studies should consider measuring exercise performance in live race scenarios, such as
overland 5-km running events with performance feedback, for longer duration endurance bouts
(e.g., 10 km, half-, or full-marathon), and explore optimal dosing strategies. Additionally, future work
should determine if CHO availability is altered with pre-sleep CHO feeding by examining muscle
glycogen, and with further consideration for sex differences, as females were shown to have higher CHO
utilization than males at rest following both HGI and LGI pre-sleep supplementation. This observation
contrasts with relatively established findings, but several factors could have contributed to females
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utilizing more CHO in the morning; we would like to acknowledge that the study was not designed
to test sex differences and there were fewer female participants (n = 6) and larger studies may prove
otherwise. Females also had, on average, lower VO2peak value (49.9 ± 4.3 mL/kg/min vs. males at
59.5 ± 5.5 mL/kg/min), and lower body weight and thus higher relative CHO loading and thus fitness
level and body weight may play a role. Another consideration of this study could be the dose of CHO
that was administered; 66 g of CHO may not be enough to last the ~eight hours to the exercise trial.
Future studies should investigate different dosages, dosing approaches (e.g., g/kg), and/or timings
of nighttime CHO supplementation for next-morning endurance performance in a larger sample,
with measurements of circulating glucoregulatory hormones or muscle glycogen which could provide
greater mechanistic insight.
5. Conclusions
The present study is the first to assess the effects of pre-sleep LGI versus HGI CHO supplementation
on next-morning exercise metabolism, GID, and endurance performance in male and female endurance
athletes. The data indicate that pre-sleep supplementation with LGI, HGI, or PLA did not differ in
GID response during exercise. There were no differences between supplements for resting REE or
RER, BG, or TT performance. In a secondary analysis, there was an interaction of supplement and
sex for FAT and CHO utilization at baseline with females utilizing more CHO with the pre-sleep LGI
and HGI, which should be explored further. In this study, consuming a CHO supplement pre-sleep,
and not within a couple of hours of exercise, might reduce GID, allowing for adequate digestion and
absorption. Future studies should investigate the effect of pre-sleep CHO supplementation on the
endurance performance of the following morning.
Author Contributions: Conceptualization, M.J.O., M.D.D., and S.J.I.; methodology, M.D.D., M.J.O., S.J.I.; formal
analysis, M.D.D., E.D.B., K.R.F., R.A.B., M.M.C., N.R.M., M.J.O., S.J.I.; investigation, M.D.D., E.D.B., K.R.F., R.A.B.,
M.M.C., N.R.M., M.J.O., S.J.I.; resources, M.J.O., S.J.I.; data curation, M.D.D., E.D.B., K.R.F., R.A.B., M.M.C., N.R.M.,
M.J.O., S.J.I.; writing—original draft preparation, M.D.D., E.D.B., K.R.F., R.A.B., S.J.I.; writing—review and editing,
M.D.D., E.D.B., K.R.F., R.A.B., M.M.C., N.R.M., M.J.O., S.J.I.; visualization, M.D.D., E.D.B., K.R.F., R.A.B., M.M.C.,
N.R.M.; supervision, S.J.I., M.J.O.; project administration, S.J.I., M.J.O. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: Foremost, the authors deeply appreciate and thank all study participants. We thank UCAN
for the donation of the UCAN superstarch to MJO for this study and the Skidmore Student Opportunity Fund for
providing modest financial assistance to help with the completion of this study.
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nutrients
Review
Gut Microbiota, Probiotics and Physical Performance
in Athletes and Physically Active Individuals
Maija Marttinen *, Reeta Ala-Jaakkola, Arja Laitila and Markus J. Lehtinen
DuPont Nutrition & Biosciences, Danisco Sweeteners Oy, Sokeritehtaantie 20, 02460 Kantvik, Finland;
[email protected] (R.A.-J.); [email protected] (A.L.); [email protected] (M.J.L.)
Abstract: Among athletes, nutrition plays a key role, supporting training, performance, and post-
exercise recovery. Research has primarily focused on the effects of diet in support of an
athletic physique; however, the role played by intestinal microbiota has been much neglected.
Emerging evidence has shown an association between the intestinal microbiota composition and
physical activity, suggesting that modifications in the gut microbiota composition may contribute to
physical performance of the host. Probiotics represent a potential means for beneficially influencing
the gut microbiota composition/function but can also impact the overall health of the host. In this
review, we provide an overview of the existing studies that have examined the reciprocal interactions
between physical activity and gut microbiota. We further evaluate the clinical evidence that supports
the effects of probiotics on physical performance, post-exercise recovery, and cognitive outcomes
among athletes. In addition, we discuss the mechanisms of action through which probiotics affect
exercise outcomes. In summary, beneficial microbes, including probiotics, may promote health
in athletes and enhance physical performance and exercise capacity. Furthermore, high-quality
clinical studies, with adequate power, remain necessary to uncover the roles that are played by gut
microbiota populations and probiotics in physical performance and the modes of action behind their
potential benefits.
Keywords: gut microbiota; probiotics; athletes; exercise; physical activity; physical performance;
cognitive performance; recovery
1. Introduction
The human gastrointestinal (GI) tract harbors a vast number of microbial cells (1014 ),
which surpasses the number of cells that make up the human body [1]. Although many intestinal
microbiota species are beneficial, others are potentially detrimental, or their functions remain unknown.
These resident microbes are involved in many metabolic processes, such as the fermentation of
undigested carbohydrates into short-chain fatty acids (SCFAs), lipid metabolism, and vitamin synthesis.
Intestinal microbiota also stimulates the maturation of the immune system and protects against
potentially pathogenic microbes [2]. Further, the microbiota may play a role in cognitive performance
and stress tolerance [3,4].
A healthy adult gut is characterized by a high degree of microbial richness (diversity) [5],
favoring health-promoting species, and features an intact epithelial barrier, which affects the
inflammatory status and nutrient utilization of the host [6]. Genetic and environmental factors,
in addition to diet and antibiotic use, have major influences on the gut microbiota composition,
starting in early childhood and extending into adulthood [7]. Dysbiosis and the loss of diversity among
gut microbiota species have been associated with various immune-regulated pathological conditions
and diseases and may, in part, contribute to the risks of developing obesity-related disorders [7,8].
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Gut microbiota populations with high degrees of microbial diversity have been associated with various
health benefits in adults. Gut microbes have the potential to exert effects via metabolites, such as
SCFAs and neurotransmitters, that can influence mucosal tissues locally or enter the circulation to
affect extra-intestinal tissues. Recently, these findings have resulted in the conceptualization of a
gut-brain axis (for review see [9]) and a gut-muscle axis (for review see [10]) indicating the existence of
bidirectional communications between the gut microbiota and the peripheral tissues of the host.
Exercise has well-known effects on cardiorespiratory fitness, muscle strength, glucose metabolism,
the immune system, and mental health [11]. Emerging evidence has indicated a plausible association
between physical activity and the gut microbiota composition [12–14]. The particular features of gut
microbiota compositions found in athletic individuals and the impacts of exercise on the gut microbiota
compositions of sedentary populations have begun to be revealed. Intervention studies have supported
the beneficial impacts of exercise and physical activity on the gut microbiota [15–17]. Furthermore,
a growing interest has developed regarding whether the modification of the gut microbiota composition
can affect the exercise and training outcomes of the host.
Probiotics are, by definition, “live micro-organisms that, when administered in adequate amounts,
confer a health benefit on the host” [18]. Probiotic supplementation may modify the gut microbiota
composition, promoting increased microbial diversity and supporting the growth of health-promoting
species [19–21]. Probiotics may also help restore a disturbed gut microbiota [15] and support a
microbiota under stress [22,23]. Although, many probiotics can support a general healthy GI and
immune system function, the specific mechanisms underlying probiotic actions, such as the production
of bioactive compounds, the inhibition of pathogen adhesion, the improvement of gut barrier function,
and immune modulation, may be highly strain-specific, even within a single bacterial species [18].
Thus far, probiotic research has primarily focused on GI function and immune regulation;
however, recent studies have targeted new research areas, such as metabolic and cognitive health.
The well-established probiotic effects on gut health and immune system function may benefit
endurance athletes, who train and perform at high intensities and often encounter physiological
challenges associated with GI and immune health during and after a competition. Therefore, probiotic
supplementation may indirectly improve the performance of an athlete by increasing the number of
healthy training and competition days and maybe even benefit stamina. The benefits of probiotics
for sports performance and training have been recognized, although the number of studies that have
examined these issues remains limited. Recently, the International Society of Sports Nutrition (ISSN)
provided a position stand on probiotics, concluding that probiotics have strain-specific effects in
athletes [24]. In this review, we provide an overview of the current research on the relationships
between exercise and gut microbiota and further evaluate the indirect and direct effects of probiotics
on physical performance, in animal models and human subjects.
2. Gut Microbiota and Physical Performance

Exercise has well-known effects on metabolism and the immune system, but the effects of exercise
on the gut microbiota have been less well studied. Compared with sedentary subjects, athletes and
physically active subjects appear to have greater fecal microbial diversity and more health-associated
microbial genera, such as Akkermansia, Veillonella and Prevotella [12–14]. However, the results of these
observational studies can only confirm associations between training status and microbiota populations,
without determining causality. In addition to physical activity patterns, sedentary subjects often differ
from physically active subjects in dietary intake patterns [25], and diet has a strong impact on the gut
microbiota composition [26].
The association between exercise and the gut microbiota composition appears to be bidirectional.
Exercise intervention studies in humans have indicated that regular physical activity modulates
the gut microbial composition [15–17]. Furthermore, growing evidence from animal studies has
also suggested that the gut microbiota plays an important role in the physical performance of the
host [27–29]. The composition and metabolic activity of gut microbiota may aid in the digestion of
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dietary compounds and improve energy harvest during exercise, which could provide metabolic benefits
for an athlete during high-intensity exercise and recovery. Observational studies have demonstrated
that the metabolic activity and pathways associated with amino acid and carbohydrate metabolism are
increased among the athlete microbiome compared with those in sedentary subjects [13,14,30].
In the gut, bacteria ferment non-digestible carbohydrates, primarily into SCFAs acetate, propionate
and butyrate. Training and regular exercise have been associated with increased fecal SCFA contents
in humans [15,30], and specific SCFAs have been associated with improved physical performance in
animal studies [14,29]. Most SCFAs are absorbed from the intestinal tract and contribute to the host’s
energy metabolism [31]. Butyrate is used primarily by epithelial cells in the colon, as an energy source.
Acetate is metabolized in muscle tissue but can also cross the blood-brain barrier. Propionate can be
used as a precursor for glucose synthesis in the liver [31]. Additionally, SCFAs improve intestinal
barrier integrity, reducing local and systemic inflammation risk. Preclinical studies have strongly
suggested that SCFAs may represent key modulators of physical performance.
Notably, the host may not be the only party to benefit from the symbiotic relationship with
microbiota during exercise. A recent study suggested that lactate, produced by the host skeletal
muscles during anaerobic exercise, enters the gut lumen through circulation, providing a selective
advantage for lactate-utilizing species that reside in the colon [14]. The results from this seminal work
imply that during high-intensity exercise, the host provides fuel, in the form of lactate, for specific
bacteria, which, in turn, produce metabolites, such as propionate, that benefit the exercising host.
Current research on the interactions between the gut microbiota and physical performance is reviewed
below and summarized in Figure 1.
Figure 1. Interactions between gut microbiota and exercise.
2.1. Gut Microbiota in Athletes

Accumulating clinical evidence has suggested that exercise modifies the gut microbiota and
that the gut microbiota composition in athletes differs from that in sedentary people, with athletes
presenting with microbial populations that are enriched in health-promoting species and have greater
diversity (Table 1). Diet and specific dietary components, such as dietary fiber, have been identified as
major influencers of the gut microbiota composition [26]. In cross-sectional and longitudinal studies,
the impacts of diet on gut microbiota cannot be excluded, especially because the dietary intake of an
athlete can greatly differ from the intake of a sedentary individual, in terms of both caloric and nutrient
contents. Most of the studies in Table 1 have reported dietary intake.
A study involving Irish, male professional rugby players showed a higher α-diversity (bacterial
richness, such as how many bacterial species are identified in fecal samples) for the gut microbiota of
athletes compared with those in sedentary controls [12]. Gut microbiota diversity correlated positively
with protein consumption and plasma creatine kinase (CK) levels, a biomarker for exercise-induced
muscle damage. A higher proportion of bacteria from the Akkermansia genus was detected in rugby
players and controls with low body mass index (BMI) compared with the proportion in controls with
high BMI. Bacteroides spp. were significantly less abundant in athletes than in controls with low BMI.
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Table 1. Studies on exercise and gut microbiota conducted in athletes, physically active individuals and sedentary population.
Training Regimen, Exercise

Subjects Dietary Intake Main Results Reference
Protocol
Athletes:
Rugby players vs.
Self-reported intake by FFQ
BMI-matched sedentary In athletes, higher α-diversity and Akkermansia spp.
In athletes, higher total energy, macronutrient and fiber
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controls Habitual training and exercise abundance vs. sedentary controls. Protein intake was [12]
intake. Protein intake 22 E% in athletes, 16 E% in
n = 86, males positively correlated with microbial diversity.
low-BMI and 15 E% in high-BMI controls
Age 29 ± 4 y
Rugby players vs.
Self-reported intake by FFQ
BMI-matched sedentary In athletes, fecal SCFAs, microbial pathways for
In athletes, higher total energy, macronutrient and fiber
controls Habitual training and exercise antibiotic biosynthesis, and amino acids and [30]
intake. Protein 22 E% in athletes vs. 16 E% in low-BMI
n = 86, males carbohydrate metabolism were increased.
and 15 E% in high-BMI controls
Age 29 ± 4 y
Prevotella spp. abundance was positively correlated
with the amount of exercise and branched chain
Professional cyclists vs. amino acid and carbohydrate metabolism pathways.
amateur cyclists Dietary intake data collected by questionnaire, reported Professional cyclists had increased Methanobrevibacter
Habitual training [13]
n = 33 (22/M, 11/F) and analyzed as overall dietary patterns. smithii transcripts and upregulated genes involved in
Age 19–49 y the production of methane compared with amateur
cyclists. No correlations between overall diet and gut
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microbiota clusters.
After the intervention, higher Bacteroidetes and lower
Habitual diet by FFQ
Cross-country runners Firmicutes abundance in the protein group.
No differences in habitual dietary intake within or
n = 18, males Bifidobacterium longum was reduced after intervention
between groups, at baseline or after the intervention.
Age: Habitual endurance training in the protein group. No changes in microbiota [32]
Dietary intervention: habitual diet and whey isolate (10 g)
Control group 35.4 ± 9.0 y composition in the control group, from pre- to
+ beef hydrolysate (10 g) or maltodextrin (control) for 10
Protein group 34.9 ± 9.5 y post-intervention. No differences within or between
weeks
groups in fecal SCFA, before or after the intervention.
Bodybuilders, long-distance
Compositional differences in bodybuilders and
runners vs. sedentary subjects Self-recorded 3-day food diary
runners associated with exercise type and diet.
n = 45, males Bodybuilders had a high-protein and distance runners
Habitual training and exercise No difference in microbial diversity between groups. [33]
Age: Bodybuilders 25 ± 3 y, had a low-dietary-fiber dietary pattern. Dietary fiber
In distance runners, protein intake was negatively
distance runners 20 ± 1 y, intake was below recommendation in all groups.
correlated with microbial diversity.
sedentary 26 ± 2 y
Self-reported intake (FFQ), detailed daily record pre-race
Highly trained ultra-endurance and during the race After the race, increased diversity and
rowers No fresh produce consumed during race. Pre-race fiber butyrate-producing species including
ca. 5000 km rowing race over 34 days [34]
n = 4, males intake: 21.45 g/day, intra-race 23.1 g/day. Only small Roseburia hominis and changes in microbial
Age 26.5 ± 1.3 y changes in intra-race macronutrient intake compared composition were observed.
with pre-race
Table 1. Cont.

Protocol
At baseline, microbiota profiles could be separated
Dietary intervention for 3 weeks with planned and into Prevotella- or Bacteroides-dominating enterotypes.
individualized menus. Subjects allocated into HCHO and PCHO resulted in minor changes, whereas
Elite race walkers
3-week structured program of High-carbohydrate diet (HCHO) LCHF resulted in stronger changes in microbial
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n = 21, males [35]

intensified training Periodized-carbohydrate diet (PCHO), or composition. LCHF was associated with reduced
Age 20–35 y
Low-carbohydrate, high-fat diet (LCHF) (ketogenic) Faecalibacterium, Bifidobacterium, and Veillonella spp.
group Increased Bacteroides and Dorea spp. in the LCHF
group was associated with decreased performance.
Marathon runners:
In marathon runners, the relative abundance of
n = 15 (4/M, 11/F)
Veillonella spp. increased post-marathon.
Mean age 27.1 y;
In ultramarathon and rower athletes, the relative
Non-runners: Habitual training and a marathon
abundance of the methylmalonyl-CoA pathway
n = 11 (5/M, 6/F) Type of exercise not reported for the
Dietary intake data collected by questionnaire (degrading lactate into propionate) in the gut [14]
Mean age 29.2 y; cohort of ultra-marathon and rower
microbiome increased post-exercise. No correlations
Ultramarathon and athletes
between dairy, protein, grains, fruits, or vegetables
rower athletes:
and Veillonella spp. abundance was observed among
n = 11 (5/M, 6/F)
marathon runners.
Age not reported
Non-athletes and sedentary subjects:
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Healthy subjects VO2 Peak test to assess CRF and to 24-h dietary recall interview
CRF correlated with microbial diversity and
n = 39 (22/M, 17/F) allocate subjects into groups (low, No significant differences in dietary intake [36]
butyrate production.
Age 18–35 y average, and high CRF) between groups.
Active vs. sedentary women
n = 40 Higher abundance of Faecalibacterium prausnitzii,
Self-reported food intake (FFQ)
Active: 30.7 ± 5.9 y, Habitual physical activity measured Roseburia hominis and Akkermansia muciniphila in active
Fiber, fruit, and vegetable intake significantly higher in [37]
BMI 24.4 ± 4.5 kg/m2 ; by accelerometer. women. Physical activity was not associated with
the active group.
Sedentary: 32.2 ± 8.7 y, differences in microbiota richness.
BMI 22.9 ± 3.0 kg/m2
Lean and obese sedentary
At baseline, the composition of gut microbiota
subjects
Exercise intervention study: 6 weeks differed between lean and obese subjects, but after
n = 32 Maintenance of habitual diet during the intervention.
of moderate-to-vigorous intensity exercise training, no difference
Lean: n = 18 (9/M, 9/F), mean A designed 3-day food menu, based on previous reported [15]
aerobic exercise and 6 weeks was observed between lean and obese subjects.
age 25.10 y; habitual diet, before fecal sample collection.
without exercise Exercise increased fecal SCFA and SCFA producing
Obese: n = 14 (3/M, 11/F), mean
bacteria in lean subjects.
age 31.14 y
Table 1. Cont.

Protocol
Gut microbiota composition was affected by BMI,
Children and teenagers
exercise frequency, and diet type. Firmicutes were
n = 267 (178/M, 89/F) Self-reported physical activity Type of diet reported as omnivore or vegetarian. [38]
significantly enriched in subjects with more
Age 7–18 y
frequent exercise.
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Habitual physical activity. Habitual diet Exercise did not affect α-diversity. Exercise increased
Overweight sedentary women
Exercise intervention study: 6-week Self-reported 3-day food record Akkermansia spp. and reduced Proteobacteria
n = 17
control period without exercise, No changes in intake of total energy, macronutrients or abundance. No significant changes in BMI or total fat [16]
Age 36.8 ± 3.9 y
6-week programmed endurance fiber from baseline, after control or exercise period. mass after exercise. Significant reduction in android
BMI 31.8 ± 4.4 kg/m2
exercise, on a bicycle ergometer A modest increase in energy from starch fat mass.
Healthy subjects CRF correlated with Firmicutes/Bacteroidetes ratio.
n = 37 (20/M, 17/F) VO2max test to assess CRF Habitual diet recorded for 7 days No correlation between dietary factors or BMI and [39]
Age 25.7 ± 2.2 y Firmicutes/Bacteroidetes ratio.
Elderly community-dwelling Habitual physical activity, measured Physical activity was not associated with α-diversity
men by activity sensor, for 5 days. Step Self-reported food intake (FFQ) but was positively associated with β-diversity.
[40]
n = 373 count as primary physical activity Step count was not associated with food or alcohol intake. Increased physical activity was associated with greater
Age 78–98 y variable Faecalibacterium and Lachnospira spp. prevalence.
Brisk walking increased the relative abundance of
Exercise intervention study:
Elderly sedentary women Self-reported food intake (FFQ) Bacteroides spp. Bacteroides spp. abundance was
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resistance training (trunk muscles) or
n = 29 No changes in energy or nutrient intake after positively associated with improved CRF after aerobic [17]
aerobic exercise (brisk walking) for
Age 65–77 y interventions. training but not with improved CRF after
12 weeks
resistance training.
BMI, body mass index; y, years; FFQ, food frequency questionnaire; E%, percentage of total energy intake; SCFA, short-chain fatty acid; M, males; F, females; VO2Peak /VO2Max , maximum
rate of oxygen consumption; CRF, cardiorespiratory fitness.
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Akkermansia sp. has been shown to inversely correlate with obesity [41] and Bacteroides spp. has
been associated with a “Western” type of diet, with high protein and fat contents [42].
Differences between rugby players and sedentary controls were also detected in the microbial
metabolism level, with increased amino acid and carbohydrate metabolism pathway activity detected
in athletes [30]. Furthermore, higher fecal SCFA (acetate, propionate, and butyrate) levels were
detected in rugby players compared with those in sedentary controls. SCFAs produced by gut
bacteria have well-known health-promoting effects on the maintenance of intestinal barrier function,
immune modulation, and the host’s energy metabolism [43,44].
Similar to Clarke et al. [12], Petersen et al. [13] reported lower levels of Bacteroides spp. in
competitive cyclists. Cyclists who trained >11 h/week had a higher relative abundance of Prevotella spp.
than those who trained less often. In addition, a meta-transcriptomics analysis showed that Prevotella
transcripts were positively correlated with branched-chain amino acid (BCAA) metabolism pathways
in the microbiome. BCAAs, especially leucine, are essential amino acids that promote muscle protein
synthesis and may enhance recovery after exercise. Further, more fecal Methanobrevibacter smithii
transcripts were identified in professional cyclists compared with amateur cyclists. M. smithii was
associated with upregulated methane metabolism, which correlated positively with upregulation
of SCFA metabolism pathways in the gut microbiome [13]. However, the authors recognized the
lack of dietary control and the absence of a non-athlete control group in the study. In line with the
results observed in cyclists, fecal microbiotas were classified into Prevotella- or Bacteroides-dominant
enterotypes in a small group of elite race walkers [35].
Scheiman et al. [14] demonstrated that the relative abundance of Veillonella spp. bacteria among
marathon runners was significantly higher after the marathon, compared with the pre-exercise
abundance. In addition, the same research group conducted metagenomic analyses using fecal
samples from ultramarathoners and Olympic level rowers, which revealed the enrichment of genes
associated with lactate and propionate metabolism in post-exercise compared with pre-exercise
samples. A follow-up study, conducted in mice, demonstrated that treatment with a Veillonella sp.
strain, which was isolated from a marathon runner, increased the treadmill running time of mice by
13% [14].
The chronological impact of prolonged, very-high-intensity exercise on the gut microbial
composition was investigated in four well-trained men who participated in a trans-oceanic rowing
competition [34]. All, except one rower, who required antibiotic treatment before mid-race,
showed increased microbial α-diversity at mid-race, which continued until the end of the race.
Baseline diversity was partially or completely restored three months after the competition. Although this
study represents a very small sample size, the microbial metabolic pathways related to specific amino
acids and medium and long-chain fatty acids tended to increase [34]. However, the diet differed
considerably during the rowing race compared with the pre-race diet; therefore, dietary change may
have also contributed to the microbial diversity findings.
In addition to the high-intense training that is practiced by professional or competitive athletes,
exercise that is performed at the recommended minimum level, based on the World Health Organization
(WHO) guidelines of 150 min of moderate-intensity exercise each week [45], appears to sufficiently
modify the gut microbiota composition [37]. Premenopausal women who practiced continuous exercise
at a low dose demonstrated increased abundance of Akkermansia muciniphila, Faecalibacterium prausnitzii,
and Roseburia hominis, compared with those in sedentary women [37]. These all are bacterial species that
are associated with health-promoting and anti-inflammatory effects [43]. Moreover, Faecalibacterium
spp. and Roseburia spp. are among the most abundant butyrate-producers in the human gut [43,44].
Different dietary patterns between physically active and sedentary groups may have influenced the
gut microbiota composition, as the intake of dietary fiber was significantly higher in active women
compared with sedentary women (mean intake 30.9 g vs. 21.4 g), and the intake of processed meat was
significantly higher in the sedentary group [37].
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Associations between physical activity levels and gut microbiota compositions have also been
demonstrated in children [38] and seniors [40]. In a study cohort of children, aged 7–18 years, from the
American Gut Project, BMI, exercise frequency, and type of diet were individually associated with the
gut microbiota composition, after controlling for covariates (age, gender, and the use of antibiotics
and probiotics) [38]. Exercise frequency was associated with gut microbiota enriched with Firmicutes
phylum. Furthermore, children who exercised daily showed an increase in genera within Clostridiales,
Lachnospiraceae, and Erysipelotrichaceae. In older men, physical activity, measured based on step
count and self-reported activity, was not associated with microbial α-diversity, but modest associations
between physical activity level and Faecalibacterium spp. and Lachnospira spp. were found [40].
These studies indicated the existence of differences in the gut microbiota composition between athletes
or physically active populations and sedentary populations. However, some of the characteristics of the
microbiota composition in athletes and physically active people may be explained by diet, rather than
the effects of exercise. Athletes often follow strict diets that support training and performance,
and exercise extremes are often associated with dietary extremes [12]. Protein supplements are often
consumed to meet the higher protein requirements of training individuals, although the popularity
of protein supplements is likely also influenced by claims regarding increased muscle mass and
improved performance and recovery [46]. Thus, protein intake can be substantially higher among
athletes compared with the normal population. Following high protein intake, unabsorbed protein
enters the colon and promotes the growth and selection of specific bacteria. Protein supplementation
(whey isolate and beef hydrolysate) for 10 weeks increased the abundance of Bacteroidetes and
decreased health-related taxa, including Roseburia spp., Blautia spp., and Bifidobacterium longum,
in runners [32]. However, the long-term effects of such alterations in the gut microbiota composition
on host health remain unclear.
Differences in dietary intake between study populations may explain some of the inconsistencies
observed among the results of different studies. In a clinical study in Korea, total protein intake
was inversely correlated with microbial diversity [33], whereas high protein intake was associated
with increased microbial diversity among Irish professional rugby players [12]. Korean athletes
did not meet the dietary recommendations for dietary fiber intake (recommendation ≥ 25 g/day;
median intake in bodybuilders 19 g/day, endurance athletes 17 g/day), whereas Irish rugby players
had fiber intake values at the recommended level (median intake 39 g/day). Undigested dietary fiber is
an important energy and carbon source for the gut microbiota, acting as a substrate for SCFA synthesis,
and representing a key contributor to microbial diversity. A high-protein diet, in combination with
low-dietary-fiber diet, may be harmful for the gut microbiota composition, rather than high protein
intake alone [47].
Limited data, derived primarily from animal studies, have suggested that popular sports nutrition
supplements, such as caffeine, BCAAs, sodium bicarbonate, and carnitine, can modify the gut
microbiota composition [48]. The effects of sports nutrition supplements on the gut microbiota remain
understudied among athletes.
To summarize, exercise and training have been associated with compositional changes in the gut
microbiota, including increased microbial diversity and increased abundance of health-promoting
microbial species. Results from large study cohorts with recreationally active subjects suggest
that exercise is associated with increases in genera within Clostridiales and Lachnospiraceae [38,40].
Although several studies have investigated small populations that likely lack sufficient statistical power,
it is intriguing that they commonly identify genera such as Akkermansia [12,37] and Prevotella [12,13] at
higher abundance in athletes and physically active subjects. However, because the number of clinical
studies remains limited, with highly different participant demographics and dietary intake—dietary
fiber intake in specific—conclusions should be drawn carefully. Observational studies that have
compared trained athletes and physically active subjects with sedentary subjects have suggested
long-term effects of exercise training on gut microbiota composition, wherein the diet plays an important
role. Sedentary and physically active subjects differ not only in their exercise patterns but also in
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their dietary intake and body composition, which are both factors that are associated with the gut
microbiota composition.
2.2. Impacts of Exercise Interventions on Gut Microbiota

Because athletes often adhere to special diets that may influence the gut microbiota, exercise
intervention studies can provide a more diet-independent approach for examining whether exercise has
an impact on the host gut microbiota (Table 1). A research group demonstrated that exercise training
intervention modified the gut microbiota composition of sedentary, non-trained, Finnish women,
without changes in dietary habits, weight, or body composition [16]. The authors demonstrated that
endurance exercise altered the gut microbiome of overweight, sedentary women, who participated in
an exercise intervention that consisted of performing a bicycle ergometer routine, three times a week,
for six weeks. The study showed no differences in total energy intake or the intake of macronutrients or
dietary fiber after the training intervention. Differences were not found in the gut microbiota α-diversity
or phylum-level abundance between pre- and post-intervention samples; however, endurance exercise
increased relative abundance of members of the genera Verrucomicrobia and Akkermansia and decreased
the number of inflammation-associated Proteobacteria in the gut. Changes in Akkermansia spp. and
genera and species within phyla Proteobacteria and Verrucomicrobia were responsive to exercise and
were independent of age, weight, percent body fat, and food intake. Another study, performed by
Morita et al. [17] found that a 12-week aerobic exercise training program significantly increased the
relative abundance of Bacteroides spp. in elderly, sedentary women, without changes in nutrient intake.
A study by Allen et al. [15] supported these findings, showing that aerobic exercise induced
changes in the gut microbiota composition, independent of dietary intake, among sedentary subjects;
however, BMI may influence the response of gut microbiota to exercise. In their study, obese and lean
individuals had different gut microbiota compositions at baseline, but after a 6-week aerobic exercise
training program, no difference was found in microbiota community composition between obese and
lean. In addition, aerobic exercise increased fecal SCFA concentrations and SCFA production capacity
in lean subjects. The effects of exercise on gut microbiota were reversed after training was discontinued.
Overall, aerobic exercise training improves cardiorespiratory fitness (CRF), an effect that has been
demonstrated in studies by Munukka et al. [16], Allen et al. [15] and Morita et al. [17]. CRF, which was
measured as the maximum rate of oxygen consumption (VO2max ), has been observed to correlate
with gut microbial diversity, fecal butyrate levels [36], and the Firmicutes-Bacteroidetes ratio [39].
The ratio between Firmicutes and Bacteroidetes phyla has been reported to be associated with body
composition, with a higher fraction of Bacteroidetes associated with higher proportions of lean body
mass, whereas lower levels have been associated with obesity [49].
In addition to human clinical studies, preclinical research in animal models has demonstrated that
exercise changes the gut microbiota composition [50–55] and fecal SCFA concentrations, by increasing
the production of butyrate [50,54], in particular. However, forced exercise, under stressful conditions,
such as the exhaustive swimming test, may impact gut microbiota differently than voluntary activity,
such as wheel running. In an overtraining mouse model, the gut microbial diversity was reduced in
mice forced to swim to exhaustion compared with that in non-swimming mice [56].
2.3. Effects of Targeted Gut Microbiota Modulation on Physical Performance

Due to nutritional, genetic, and environmental factors, dissecting the exact role played by gut
microbiota on exercise performance in human clinical studies can be difficult. Germ-free animal
models overcome many of those challenges and have been used to demonstrate the roles played by
gut microbiota on physical performance outcomes. Hsu et al. [27] studied the swimming capacities of
specific pathogen-free (SPF), germ-free (GF), and Bacteroides fragilis gnotobiotic mice. The swim-to-
exhaustion time was the shortest for GF mice and the longest for SPF mice, indicating decreased
performance in the absence of gut microbiota. Similar findings regarding the reduced performance of
GF mice compared with that in gnotobiotic and SPF mice were observed by Huang et al. [57].
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In contrast to the above, Lahiri et al. [58] showed that GF mice and SPF mice did not differ in physical
performance when exercising until exhaustion. However, GF mice demonstrated reduced muscle
mass, fewer muscle fibers, and reduced muscle strength compared with SPF mice. Muscle atrophy in
GF mice was associated with dysregulated mitochondrial biogenesis and reduced oxidative capacity.
The transplantation of gut microbiota from SPF mice restored the muscle mass in GF mice, and treatment
with a blend of SCFAs increased skeletal muscle mass and muscle strength in GF mice compared with
those in untreated GF mice [58].
Antibiotic treatment drastically alters the composition of gut microbiota. Nay et al. [28]
demonstrated that gut microbiota depletion, following a broad-spectrum antibiotic treatment,
reduced the endurance running time of mice, and the endurance capacity was normalized after
microbiota restoration through reseeding. Changes in endurance capacity were not related to changes
in muscle mass, muscle fiber typology, or mitochondrial function but were associated with changes
in muscle glycogen levels, which were restored after reseeding. Okamoto et al. [29] reported similar
findings, in which the treadmill running time was shorter in mice treated with multiple antibiotics
compared with that in non-treated controls. Okamoto et al. [29] also investigated the effects of SCFA
production and its role on exercise performance, by feeding mice with fibers with differential substrate
availability for microbial SCFA production in the gut. Mice fed with reduced fermentable fibers showed
significantly shorter running times compared with mice fed with highly fermentable fibers, suggesting
that microbiota and its substrates are both associated with physical performance. To further explore
the putative role of SCFAs in performance capacity, antibiotic-treated mice were administered with a
subcutaneous infusion of acetate or butyrate [29]. Acetate, but not butyrate, infusion improved the
antibiotic-induced deterioration in running time.
Germ-free animals are of course an extreme model and may not explain the more subtle difference
observed in the microbiota of humans. Nevertheless, studies in germ-free animal models have
established a cause-effect relationship between gut microbiota and physical performance. Overall,
the normalization of gut microbiota dysbiosis appeared to effectively restore exercise capacity and
skeletal muscle parameters in rodents [58]. In addition, differences in gut microbiota compositions
or the lack of gut microbiota have been shown to modulate exercise capacity, associated with muscle
structure, muscle strength, and/or energy utilization [25,28]. Thus, the host appears to benefit from
microbes through improved performance. The effects of gut microbiota are at least partially mediated
by the production of SCFAs, which impact the gut and can also affect peripheral target tissues,
via circulation.
3. Probiotics as a Potential Ergogenic Aid to Enhance Physical Performance

Nutritional ergogenic aids are dietary supplements that are consumed to help an individual
exercise, enhance exercise performance capacity, enhance training adaptations, and improve recovery
from exercise [59]. The use of nutritional ergogenic supplements is popular among athletes and
recreationally active individuals of all age groups; however, evidence that supports the efficacy of many
supplements is very limited or lacking. Performance-enhancing supplements with good or strong
evidence have been identified by the International Olympic Committee and include the following:
caffeine, creatine, nitrate, sodium bicarbonate, and beta-alanine [60]. Even when associated with only
marginal improvements in performance, safe, proven, ergogenic dietary supplements may provide
competitive benefits for an athlete.
Probiotic supplementation has been demonstrated to beneficially modify and support the gut
microbiota composition [19–21]. Probiotics comprise many bacterial species, with the most studied
probiotics belonging to the genera Lactobacillus (and associated genera) or Bifidobacterium. Associations
between probiotics and physical performance and plausible mechanisms underlying these actions have
been addressed in animal studies, which have suggested that probiotic supplementation protects against
undesirable physiological changes that may be induced by strenuous exercise. Preclinical studies have
demonstrated that probiotics can improve gut barrier properties [61] and the antioxidative status [62]
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and attenuate inflammatory response [63–65] in rodents after exhaustive exercise. However, how these
protective effects are associated with physical performance outcomes has not been determined.
To date, the effects of probiotic supplementation have been studied in a variety of athletic
and physically active populations, examining a variety of probiotic strains. Because the number of
clinical studies on the association between probiotics and physical performance remains very low,
with each study generally including a small number of participants and utilizing different exercise
protocols, conclusions should be drawn carefully. The training status and training history of the
participants can also influence the outcomes of exercise interventions [66]. Trained athletes and
untrained individuals differ in their physiological responses to exercise [66], which can result in
controversial results among different study populations. The use of resistance training programs alone
during exercise intervention studies can contribute to changes in body composition and skeletal muscle
organization that may supersede the impacts associated with probiotic supplementation, especially
among previously sedentary populations.
The studies that have examined the effects of probiotics on physical performance have generally
focused on mid- to long-term benefits, with supplementation periods varying from 2 weeks to 3 months
(Table 2). The examined probiotic strains, formulas, and doses vary from study to study, which creates
controversy among the obtained results. The most studied species are members of genera Lactobacillus
(and associated genera) and Bifidobacterium; however, the benefits of probiotics, even within a single
species, are often strain-specific. Furthermore, studies have been performed using both live and
inactivated bacteria, which may have different modes of action. To comply with the definition,
probiotics need to be alive microbes [18]. The proposed mode-of-action for probiotics and the benefits
they provide to athletes are summarized in Figure 2.
Figure 2. Proposed mechanisms and benefits of probiotic use in athletes.
3.1. Reduction of Gastrointestinal and Upper Respiratory Tract Symptoms

The beneficial effects of probiotics on GI health and upper respiratory tract (URT) illness symptoms
among the general population have been well-acknowledged and reviewed extensively, elsewhere [67,68].
Because the exercise performance capacity of an athlete can be greatly influenced by overall health and
resistance to infections, we have briefly highlighted the main findings, here.
Several studies have shown the potential of probiotic use to shorten the duration of GI disturbance
episodes and relieve GI symptoms in athletes [69–74]. GI symptoms are common in athletes and can
be influenced by the type of exercise performed [75]. GI challenges are most prominent in endurance
athletes, among whom the prevalence of symptoms varies from 30% to up to 90%, depending on the
individual athlete and the type and the extremes of the exercise [75]. Typically, endurance athletes
suffer from mild to severe symptoms, including nausea, vomiting, abdominal angina, and bloody
diarrhea, which are caused, in part, by reduced blood circulation in the splanchnic region during
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intensive exercise [75]. Reduced circulation can result in oxygen deprivation in the gut epithelial
cells, which damages the cells and causes changes in the gut permeability, a phenomenon known as
“leaky gut syndrome.” Metabolites, such as SCFAs, and other effector molecules, which are produced
by beneficial bacteria, may improve the intestinal barrier function by increasing the expression of tight
junction proteins in the epithelia, which reduces mucosal permeability [43]. Clinical results regarding
the effects of probiotic administration on gut permeability in athletes are scarce and controversial,
showing positive effects [72,73] or no effects [76,77].
Prolonged high-intensity exercise has been associated with transient immune dysfunction and
increased illness risk [78]. Due to the transient suppression of mucosal and systemic immune responses,
athletes are especially susceptible to viral respiratory infections, which affect the quality of training
and physical performance [79,80]. In contrast, moderate exercise appears to protect against infections,
whereas a sedentary lifestyle increases the risk [78,79]. Several studies have investigated whether
probiotic supplementation can reduce the risks of respiratory tract illness episodes, alleviate symptoms,
and reduce the duration of episodes among athletes and recreationally active subjects. The study results
have not shown consistent effects for all of the aforementioned benefits; however, beneficial effects
on incidence, duration, and the number of symptoms have been reported [69,81–85]. Consequently,
the positive impacts of probiotic supplementation on URT symptoms and illness may facilitate an
earlier return to normal activity levels, references [83,85] increasing the hours spent on training,
which can positively influence overall athletic performance. The administration of a two-strain
probiotic supplement (Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bi-07)
delayed the occurrence of URT illness and significantly increased the training load during a 5-month
intervention period compared with placebo [83].
Reducing the incidence or severity of illness has positive impacts on performance during training
and competition. Thus, probiotics may indirectly enhance physical performance.
3.2. Enhancement of Physical Performance

Depending on the sport and exercise type, physical performance can be measured as outcomes
related to endurance, strength, speed, flexibility, or psychological performance (concentration,
motivation) [86]. Exercise capacity often refers to exercise time to fatigue or exhaustion, at a given
intensity or workload [87]. The potential for probiotics to improve physical performance has been
recognized during exercise interventions and training studies involving athletes, recreational athletes,
and sedentary individuals. Table 2 summarizes the studies examining the associations between
physical performance and probiotic use, including preclinical and clinical studies, in which the used
supplementation protocol fulfills the definition of a probiotic as a live organism.
Probiotic supplementation has been shown to increase the time to fatigue in both preclinical
studies [88–90] and clinical studies, among both athletes and non-athletes [77,91,92]. Lactiplantibacillus
plantarum TWK10 is among the most studied probiotic strains in terms of physical performance outcomes.
A preclinical animal study demonstrated a dose-dependent increase in forelimb grip strength and
endurance swimming time in mice supplemented with TWK10 [88]. Mice supplemented with TWK10
also showed an increased number of type I (slow-twitch) muscle fibers in the gastrocnemius muscle
compared with control mice. These performance benefits were further confirmed in clinical studies.
Endurance performance in an exhaustive treadmill exercise was improved in healthy, untrained adult
males, who were supplemented daily with TWK10 for 6 weeks, compared with those who received
a placebo [91]. In addition to significantly longer time to exhaustion (58% longer running time in
the probiotic vs. placebo groups), the post-exercise blood glucose level was higher in TWK10 group
compared with the placebo group suggesting improved energy harvest from gluconeogenic precursors
during exhaustive exercise. No significant improvements in perceived exertion during exhaustive
exercise were reported by the probiotic supplementation group compared with the placebo group.
A more recent clinical study from the same research group demonstrated a dose-dependent
improvement in endurance performance (time to exhaustion) following TWK10 supplementation
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(3 × 1010 or 9 × 1010 colony forming units, CFU, per day) in untrained subjects [92]. A higher dose
of TWK10 significantly increased muscle mass compared with placebo treatment during the 6-week
supplementation period. Further, blood lactate levels were significantly lower at the end of the exercise
bout after both doses of probiotic supplementation compared with placebo treatment.
A double-blind, cross-over, exercise study examining trained male runners demonstrated
that supplementation with a multi-strain probiotic (L. acidophilus, Lacticaseibacillus rhamnosus,
Lacticaseibacillus casei, L. plantarum, Limosilactobacillus fermentum, Bifidobacterium lactis, B. breve, B. bifidum,
and Streptococcus thermophilus) for 4 weeks significantly increased the time to fatigue on a treadmill
running exercise performed in the heat compared with placebo, resulting in an average 16% longer
running time [77]. No differences were observed in the severity of GI symptoms or GI permeability
between the probiotic and placebo groups during exercise [77].
However, not all studies have shown enhancements in endurance performance following probiotic
use in highly trained subjects or athletes [81,84,85,93]. Performance measurements related to exhaustive
endurance exercise were not affected in endurance-trained males, after 4 weeks of L. fermentum VRI-003
supplementation [81], or in trained subjects, after Lactobacillus helveticus Lafti L10 [84]. A multi-species
probiotic formulation (B. bifidum W23, B. lactis W51, Enterococcus faecium W54, L. acidophilus W22,
Levilactobacillus brevis W63, and Lactococcus lactis W58) for 3 months did not have benefit in endurance
performance in highly trained athletes [85]. In female swimmers, a multi-strain probiotic (L. acidophilus
SPP, L. bulgaricus, B. bifidum, and S. thermophilus) yogurt improved the VO2max (calculated using a
Harvard step test) but had no impact on the 400-m swimming time after a 2-month intervention [82].
The 6-week supplementation with B. longum 35,624 in competitive, high-level, female swimmers did
not enhance aerobic or anaerobic swimming performance or improve power or force production
measurements [93]. Marshall et al. [94] found no effects for a 12-week multistrain probiotic or
probiotic + glutamine supplementation protocol on the time to complete an ultra-marathon race
compared with controls.
A few clinical studies have addressed the impacts of probiotic supplementation on sprint
and power performance showing no clear benefits. Bacillus subtilis DE111 did not improve either
strength or performance in male [95] or female athletes [96] when combined with a training protocol
involving resistance exercises. Multi-strain probiotic supplementation for 12 weeks, combined with
circuit-training, which involved resistance exercises, improved muscular performance to a similar
degree as circuit-training alone in healthy, sedentary males [97], confirming the positive effects of
resistance training on muscular outcomes, which is a result that has been demonstrated by other
probiotic and exercise interventions among athletes [95,96]. The effects of probiotic supplementation
on muscle strength and power production may be superseded by the effects of the resistance training
protocols used by these studies. Regular resistance exercise strongly induces physiological changes in
skeletal muscles and improves muscular strength in the long term
A recent trend in the field of gut microbiota and exercise research has been to isolate gut bacteria
from the feces of elite athletes, to study the performance benefits of athlete-derived gut bacteria
in animals. Recently, the oral administration of either B. longum subsp. longum OLP-01 [98] or
Ligilactobacillus salivarius subsp. salicinius SA-03 [99], isolated from a female weightlifting Olympic
medalist, was demonstrated to significantly increase forelimb grip strength and endurance capacity
in a swim-to-exhaustion test in mice. Both OLP-01 and SA-03 significantly decreased blood lactate,
ammonia, and CK levels after an acute exercise bout, and increased hepatic and muscle glycogen
stores at autopsy and decreased which indicated improved energy utilization and the attenuation of
fatigue-related biomarkers in mice.
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The inoculation of Veillonella atypica, isolated from a marathon runner, increased the treadmill
running time in mice compared with that of control mice [14]. In a subsequent experiment,
the intracolonic infusion of propionate also improved running times until exhaustion in mice.
Veillonella species are known to metabolize lactate into propionate and acetate. Notably, a series
of experiments by this research group also showed that 13 C3 -lactate injected into the mouse tail vein
could be found in the contents of colon and cecum, post-injection, indicating that circulating lactate
can pass through the intestinal epithelium into the gut lumen. This seminal work in mice implies
that lactate that is produced by skeletal muscles during prolonged anaerobic exercise may enter the
colon from the circulation, which can serve as fuel for certain bacteria in the gut, providing a selection
advantage [14]. These findings suggested that both the host and gut microbes may benefit from a
symbiotic relationship; however, clinical evidence remains necessary to provide additional proof of
these beneficial effects. Although athlete-originating microbes, such as Veillonella, sp. may show
benefits in preclinical settings, the development of clinically proven commercial probiotics that can
provide health benefits in humans requires further research.
Thus far, the number of human clinical studies investigating the impacts of probiotics on physical
performance remains low, and those that have been performed have examined limited exercise types
and performance measures. Clinical data have suggested that probiotics may improve the time to
exhaustion during endurance exercise; however, these data are scarce and contradictory results exist.
Studies have been conducted using a variety of probiotic strains that may differ in their efficacy.
Further research remains necessary to determine the direct effects of probiotic supplementation on
performance outcomes.
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Table 2. Probiotic studies on physical performance, post-exercise recovery and cognitive outcomes.
Exercise Protocol and/or

Subjects Design Probiotic Supplementation Main Results Reference
Intervention
Animal studies:
Forelimb grip strength PRO improved forelimb grip strength and
Forced swim-to-exhaustion test, exhaustive swimming time. Blood lactate, ammonia,
6-week-old male ICR mice L. plantarum TWK10 (LP10)
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with loads and CK levels were lower in PRO mice after a 15-min
3 groups Animal study Dosing per group: 0, 2.05 × 108 ; or [88]
15-min swim test to determine swim compared with those in control mice. Type I
n = 8/group 1.03 × 109 CFU/kg/day for 6 weeks
recovery and fatigue-related muscle fiber type increased, and relative muscle
biomarkers weight increased in PRO mice vs. control mice.
A kefir drink with L. fermentum Kefir supplementation increased time-to exhaustion,
Forelimb grip strength DSM 32,784 (LF26), L. helveticus and improved forelimb grip strength.
Forced swim-to-exhaustion test with DSM 32,787 (LH43), L. paracasei Blood lactate, ammonia, blood urea nitrogen,
6-week-old male ICR mice
loads DSM 32,785 (LPC12), L. rhamnosus and CK levels were lower after exercise in kefir-fed
4 groups Animal study [89]
10-min and 90-min swim tests, DSM 32,786 (LRH10), and S. mice compared with control mice, in a
n = 8/group
to determine recovery and thermophilus DSM 32,788 (ST30) dose-dependent manner. Glycogen contents in the
fatigue-related biomarkers Kefir dosing per group: 0, 2.15, liver and muscle were higher in kefir-supplemented
4.31, or 10.76 g/kg/day for 4 weeks mice compared with control mice.
11-week-old male Wistar Incremental speed exercise on a PRO supplementation moderately improved aerobic
Saccharomyces boulardii (strain not
rats treadmill, until exhaustion performance. PRO mice ran approx. 8 min longer
Animal study reported) [90]
2 groups Treadmill chamber, coupled with than control mice (until exhaustion) and had higher
3 × 108 CFU/kg/day for 10 days
n = 13/group gas-analyzer, to assess VO2max maximal speed.
187
PRO improved forelimb grip strength and
swim-to-exhaustion time, in a dose-dependent
Forelimb grip strength
B. longum subsp. longum OLP-01 manner. Blood lactate and ammonia levels were
Forced swim-to-exhaustion test,
7-week-old male ICR mice isolated from a female weightlifter lower after the acute swim test in PRO vs. control
with loads
4 groups Animal study Dosing per group: 0, 2.05 × 109 , mice. After a 90-min swim test, blood urea nitrogen [98]
10-min and 90-min swim tests,
n = 10/group 4.10 × 109 , or 1.03 × 1010 and CK levels were lower in PRO mice compared
to determine recovery and
CFU/kg/day for 4 weeks with those in control mice. PRO increased hepatic
fatigue-related biomarkers
and muscular glycogen contents, observed at
autopsy.
PRO improved forelimb grip strength and
swim-to-exhaustion time, in a dose-dependent
Forelimb grip strength L. salivarius subsp. salicinius
manner. Blood lactate and ammonia levels were
Forced swim-to-exhaustion test, SA-03, isolated from a female
6-week-old male ICR mice lower and blood glucose levels were higher after
with loads weightlifter’s gut microbiota
4 groups Animal study acute tests in the PRO groups vs. control group. [99]
10-min and 90-min swim tests, Dosing per group: 0, 2.05 × 109 ,
n = 10/group After a 90-min swim, blood CK levels were lower in
to determine recovery and 4.10 × 109 , or 1.03 × 1010
PRO groups compared to the control group. PRO
fatigue-related biomarkers CFU/kg/day for 4 weeks
increased hepatic and muscular glycogen contents,
observed at autopsy.
Table 2. Cont.

Intervention
Clinical studies:
Swimmers
6 weeks of intensified off-season
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training, including swimming and

resistance exercise.
No significant differences in exercise performance or
Performance assessment: Vertical
Highly trained competitive systemic inflammation markers (at rest) between
Randomized, jump force plate test, aerobic and B. longum 35,624;
swimmers PRO and PLA.
double-blind, anaerobic swim performance test 1 × 109 CFU bacteria/day for [93]
n = 17, females Differences in cognitive outcomes were detected
placebo-controlled Cognitive assessment: stress and 6 weeks
Age not reported showing more favorable sport recovery related
recovery during the intensified
scores in the PRO group.
exercise training load (the
Recovery-Stress Questionnaire for
Athletes)
L. acidophilus SPP, L. delbrueckii
Normal exercise regimen subsp. bulgaricus, B. bifidum,
Swimmers Significant improvement in VO2max in the PRO
Randomized, Performance assessment: 400-m free- and S. salivarus subsp.
n = 46, females group. No differences in 400-m swimming times [82]
placebo-controlled swimming record, Harvard step test thermophilus, strains not reported
Age 13.8 ± 1.8 y between PRO and PLA groups.
to, measure VO2max 400 mL of probiotic yogurt/day
with 4 × 1010 CFU/mL for 8 weeks
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Endurance runners
Habitual winter-season training
L. fermentum VRI-003; No difference in performance outcomes with PRO
Randomized, Performance assessment: A
Elite distance runners 1.2 × 1010 CFU bacteria/day compared to PLA. The number of illness days during
double-blind, treadmill running test until
n = 20, males for 4 weeks PRO supplementation was significantly lower than [81]
placebo-controlled, exhaustion, at the start of the study
Age 27.3 ± 6.4 y Cross-over study, with 1-month with PLA (30 vs. 72 days). IFN-γ response was
crossover period and the end of each study
wash-out moderately higher with the PRO than with PLA.
month
Habitual training L. casei (strain not reported) No differences in hydration status between PRO and
Endurance-trained runners Randomized, blinded,
Bout of exercise: 2-h running 1 × 1011 CFU/day for 7 days PLA. Inflammatory cytokine levels were not
n = 8, males placebo-controlled, [100]
exercise at 60% VO2max in hot Cross-over study, with 1-month different between PRO and PLA, either pre-exercise
Age 26 ± 6 y cross-over
ambient conditions wash-out or post-exercise (1, 2, 4, and 24 h after running).
Habitual training L. casei (strain not reported) PRO and PLA did not differ in salivary
Endurance-trained runners Randomized, blinded,
Bout of exercise: 2-h running 1 × 1011 CFU/day for 7 days anti-microbial protein or serum cortisol responses
n = 8, males placebo-controlled, [101]
exercise, at 60% VO2max, in hot, Cross-over study with 1-month during the post-exercise period (1, 2, 4, and 24 h after
Age 26 ± 6 y cross-over
ambient conditions wash-out running).
Multispecies probiotic, strains not
PRO increased run time to fatigue (PRO 37:44 vs.
specified; L. acidophilus,
PLA 33:00 min:sec). A moderate, non-significant
Randomized, Normal training L. rhamnosus, L. casei, L. plantarum,
Runners reduction in pre-exercise and post-exercise serum
double-blind, Performance assessment: Running L. fermentum, B. lactis, B. breve,
n = 10, males lipopolysaccharide (LPS) levels for PRO compared to [77]
placebo-controlled, to fatigue, at 80% of ventilatory B. bifidum, and S. thermophilus
Age 27 ± 2 y PLA. No difference between PRO and PLA in
cross-over threshold, at 35 ◦ C and 40% humidity 45 × 109 CFU/day for 4 weeks,
plasma IL-6, IL-10, and IL-1Ra or GI permeability
cross-over study with a 3-week
after exercise in the heat.
wash-out
Table 2. Cont.

Intervention
PRO maintained salivary immune protection and
increased anti-inflammatory response on the upper
Marathon runners Randomized,
Usual training L. casei Shirota airways, immediately after the marathon. Serum
n = 42, males double-blind, [102]
Bout of exercise: marathon run 40 × 109 CFU/day for 30 days TNF-α level was significantly lower immediately
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Age 39.5 ± 9.4 y placebo-controlled

post-marathon in the PRO group compared to that in
the PLA group
L. rhamnosus GG
Marathon runners Randomized, 3-month training period, 6-day
4.0 × 1010 bacteria in drink/day PRO did not differ from PLA in ox-LDL or
n = 119 (105/M, 14/F) double-blind, preparation period [103]
(or 1 × 1010 in tablet/day) for 3 antioxidant activity, pre- or post-marathon.
Average age 40 y placebo-controlled Bout of exercise: marathon run
months
No difference in marathon times between PRO and
PLA. During the final third of the race, the reduction
Randomized, Habitual training routine L. acidophilus CUL60, L. acidophilus
Marathon runners in average relative speed was greater in PLA
double-blind, Performance assessment/Bout of CUL21, B. bifidum CUL20, and B.
n = 24 (20/M, 4/F) compared to PRO. GI symptoms were lower in PRO [104]
placebo- controlled, exercise: Marathon race (no baseline animalis subsp. lactis CUL34
Age 22–50 y compared to PLA during the final third. No
matched-pairs assessment) 2.5 × 1010 CFU/day for 28 days
difference in post-race serum IL-6, IL-8, IL-10,
and cortisol levels between groups.
PRO: Multi-strain probiotic, daily
dose 30 × 109 CFU comprising of
189
10 × 109 CFU L. acidophilus
CUL-60 (NCIMB 30,157), 10 × 109
CFU L. acidophillus CUL-21
(NCIMB 30,156), 9.5 × 109 CFU B.
bifidum CUL-20 (NCIMB 30,172),
and 0.5 × 109 CFU B. animalis
Training for a marathon,
subsp. lactis CUL-34 (NCIMB
ultra-marathon race of 294 km No difference in pre-race VO2max or in
30,153 + 55.8 g
Randomized, controlled Performance assessment: time-to-completion for ultra-marathon between
Ultramarathon runners fructooligosaccharides
(single-blind for A graded exercise test, to maximal PRO, PRO + glutamine, and control groups.
n = 32 (26/M, 6/F) PRO + glutamine: [94]
glutamine exhaustion, on a motorized treadmill, PRO and PRO + glutamine had no effects on
Age 23–53 y Daily dose 2 × 109 CFU L.
supplementation) VO2max test, pre-marathon, immune activation via extracellular heat-shock
acidophilus CUL-60 (NCIMB
time-to-completion in ultra- protein eHsp72 signaling at post-race.
30,157), 2 × 109 CFU L. acidophilus
marathon race
CUL-21 (NCIMB 30156), 5 × 107
CFU B. bifidum CUL-20 (NCIMB
30,172), 9.5 × 108 CFU B. animalis
subsp. lactis CUL-34 (NCIMB
30,153), and 5x 109 CFU L.
salivarius CUL61 (NCIMB 30,211)
+ 0.9 g glutamine
12 weeks before the marathon
Table 2. Cont.

Intervention
Cyclists, triathletes
Habitual training (physical activity
Competitive cyclists recorded) PRO did not affect training patterns or performance
Randomized,
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n = 99 (64/M, 35/F) Performance assessment: an L. fermentum VRI-003 PC in VO2 max testing. Acute exercise-induced changes
double-blind, [71]
Age 35 ± 9 y/M and incremental cycle ergometer 1 × 109 CFU/day for 11 weeks in anti- and pro-inflammatory cytokines were
placebo-controlled
36 ± 9 y/F performance test (peak power output, attenuated with PRO.
VO2max )
In Study II, performance during recovery from a full
triathlon was decreased in the PLA group and
maintained at the pre-triathlon level in the PRO
group. PRO group had lower blood TNF-α, IFN-γ,
IL-6, and IL-8 levels compared to PLA, immediately
8 weeks of programmed training
Triathletes after exercise (Study I/II), with levels significantly
before a sprint triathlon (Study I) or L. plantarum PS128
Study I: n = 18, Randomized, lower in PRO group 3 h after full triathlon (Study II).
full triathlon competition (Study II) 3 × 1010 CFU/day
Study II: n = 16 double-blind, Anti-inflammatory IL-10 was higher in the PRO [105]
Performance assessment: Wingate Study I: last 4 weeks of training
Sex not reported placebo-controlled group, immediately after exercise (Study II)
and 85% VO2max test (after full Study II: last 3 weeks of training
Age 19–26 y compared with that in the PLA group. No
triathlon)
differences in muscle damage or fatigue markers
detected between groups (Study I/II) except, lower
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CK in PRO vs. PLA, 3 h after full triathlon (Study II).
Oxidative stress marker (MPO) was lower in PRO
after exercise, with no differences 3 h post-exercise.
Habitual training >11 h/week,
Elite athletes (badminton,
self-reported training loads
triathlon, cycling, alpinism, No difference in VO2max and treadmill performance
Randomized, Performance assessment: VO2max ,
karate, savate, kayak, judo, L. helveticus Lafti L10 between PRO and PLA. Increase in the subjective
double-blind, by a graded cardiopulmonary test, on [84]
tennis, and swimming) 2 × 1010 CFU/day for 14 weeks feeling of vigor in the PRO group, but no difference
placebo-controlled a treadmill
n = 50 (36/M, 14/F) in other cognitive scores between groups.
Cognitive assessment: Profile of
Age 18–28 y
mood and state (POMS) questionnaire
Multistrain probiotic, daily dose
30 × 109 CFU (10 × 109 CFU
L. acidophilus CUL-60 (NCIMB
30,157), 10 × 109 CFU L.
Non-significantly faster times were reported for PRO
acidophillus CUL-21 (NCIMB
during swim and cycle stages, and a trend towards
Standardized training program for 30,156), 9.5 × 109 CFU B. bifidum
an overall faster time was reported compared to PLA
Recreational triathletes Randomized, the previous 6 months CUL-20 (NCIMB 30,172), 0.5 × 109
(~86 min faster).
n = 30 (25/M, 5/F) double-blind, Performance assessment/Bout of CFU B. animalis subsp. lactis [73]
No baseline measurements on performance
Age 35 ± 1 y placebo-controlled exercise: a long-distance triathlon (no CUL-34 (NCIMB 30,153)) + 55.8 g
were assessed.
baseline assessment) fructo-oligosaccharides, alone or
PRO reduced post-race plasma endotoxin levels,
in combination with 600 mg
whereas PLA had no effect.
N-acetyl carnitine + 400 mg
α-lipoic acid
for 12 weeks before and
6 days after triathlon
Table 2. Cont.

Intervention
Team sports
Offseason resistance training protocol
Division I volleyball and
Randomized, Performance assessment: 1RM PRO had no effect on strength or athletic
soccer athletes Bacillus subtilis DE111
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double-blind, testing (bench press, squat, deadlift), performance but significantly reduced percentage of [96]
n = 23, females 5 × 109 CFU/day for 10 weeks
placebo-controlled isometric midthigh pull, vertical jump body fat percentage.
Age 19.6 ± 1.0 y
height, pro-agility test
Resistance training program No differences between PRO and PLA in strength,
Division I baseball athletes Randomized, Performance assessment: 1RM performance, or body composition. PRO reduced
Bacillus subtilis DE111
n = 25, males double-blind, testing (squat, deadlift), pro-agility TNF-α levels, but no differences in IL-10, cortisol, [95]
1 × 109 CFU/day for 12 weeks
Age 20.1 ± 1.5 y placebo-controlled test, 10-yard sprint, standing long zonulin, or testosterone levels observed between
jump PRO and PLA.
No difference in performance between groups.
B. bifidum W23, B. lactis W51, Weekly training loads were significantly higher in
Normal training
Highly trained athletes Randomized, Enterococcus faecium W54, L. PRO compared to PLA (8.0 ± 2.3 vs. 6.6. ± 4.3
Performance assessment: Cycle
n = 29 (13/M, 16/F) double-blind, acidophilus W22, L. brevis W63, h/week). [85]
ergometer exercise test until
Age 20–35 years placebo-controlled and L. lactis W58 Exercise-induced reduction in tryptophan levels in
exhaustion
1 × 1010 CFU/day for 12 weeks PLA but not in the PRO group. PRO reduced the
incidence of URT infections.
Active non-athletes
191
PRO attenuated performance decrements caused by
Muscle-damaging eccentric exercise muscle-damaging exercise during the recovery
Randomized, S. thermophilus FP4, and
Resistance trained subjects bout period.
double-blind, B. breve BR03
n = 15, males Performance assessment: isometric No effects of PRO on muscle soreness, range of [106]
placebo-controlled, 5 × 109 CFU of each/day for 21
Age 25 ± 4 y peak torque, after muscle motion, or plasma creatine kinase. PRO lowered
crossover days
damaging-exercise resting IL-6 concentrations that were sustained until
48 h post-exercise.
PRO + casein increased perceived recovery status
and reduced muscle soreness after exercise
Single-leg exercise bout
compared with casein alone.
Single-blind, Performance assessment: Anaerobic
Recreational exercisers Bacillus coagulans BC30 PRO + casein maintained post-exercise Wingate
crossover (casein first, power by modified Wingate test,
n = 29, males 1 × 109 CFU/day + 20 g casein for peak power at the pre-exercise level, whereas casein [107]
after washout, single-leg vertical jump, strength, by
Age 21.5 ± 2.8 y 14 days alone demonstrated reduced post-exercise
PRO+casein) 1RM testing in the one-legged leg
performance. For 1RM leg-press and vertical jump
press, after muscle damaging-exercise
power, no differences between groups in
post-exercise performance.
No difference in VO2max between PRO and PLA.
Habitual moderate exercise PRO yogurt increased antioxidant enzyme activities
Physically active subjects Probiotic not specified
Performance assessment: treadmill and reduced MMP2 and MMP9 levels before and
n = 27, females Controlled, randomized 450 g of probiotic yogurt/day for [108]
running until exhaustion, VO2max test after exhaustive exercise. No significant differences
Age 18–25 y 2 weeks
(Bruce test) between PRO and PLA in high-sensitivity CRP, IL-6,
and TNF-α after intense exercise.
Table 2. Cont.

Intervention
L. acidophilus, L. delbrueckii subsp.
bulgaricus, Lactococcus lactis subsp.
lactis, L. casei, L. helveticus, L.
Habitual training including plantarum, L. rhamnosus, L. Rating of perceived exertion during exercise was not
Nutrients 2020, 12, 2936
Physically active students

endurance exercise salivarius subsp. salivarius, B. breve, different between PRO and PLA. PRO did not affect
n = 11, sex not reported Non-controlled [109]
Bout of exercise: 2-h cycling at 60% B. bifidum, B. infantis, B. longum, salivary antimicrobial proteins at rest or in response
Age 22 ± 1 y
of VO2max Bacillus subtilis, S. thermophilus to an acute bout of prolonged exercise.
minimum 2 × 109 CFU/capsule,
3 capsules/day
for 30 days
The exercise groups completed
structured, long-distance, endurance
Students
run training, whereas the active Probiotic kefir, probiotic strain No effect of PRO on 1.5-mile completion time.
n = 67, males and females
Controlled group maintained their usual exercise and dose not specified PRO attenuated exercise-induced inflammation, [110]
(n not specified by sex)
routine. 15 weeks measured as serum CRP levels.
Age 18–24 y
Performance assessment: 1.5-mile
(2.41 km) walk or run
Students of physical Habitual training and training Probiotic strains unspecified,
education program by the study included S. thermophilus and/or PRO improved VO2max and aerobic performance.
Randomized, matched
192
n = 30, males Performance assessment: Cooper L. delbrueckii subsp. bulgaricus PRO decreased serum high-sensitivity CRP and [111]
pairs
Average age: PRO 21.56 y, test, maximum aerobic power, using 1 × 105 CFU/g in 200 mL increased HDL levels.
PLA 21.28 y Bulk test on a laboratory treadmill yogurt/day for 10 weeks
PRO improved time-to-exhaustion (PLA vs. PRO:
Habitual exercise
Healthy participants Randomized, 817 ± 79 s vs. 1292 ± 204 s). Blood glucose was
Performance assessment: Treadmill L. plantarum TWK10
n = 16, males double-blind, higher in PRO vs. PLA after exhaustive exercise. No [91]
running at 85% VO2max workload, 1 × 1011 CFU/day for 6 weeks
Age 20–40 y placebo-controlled differences in post-exercise blood lactate, free fatty
until exhaustion.
acid, CK levels between PRO and PLA.
Exhaustion time was increased in both PRO groups
Habitual exercise and were longer compared to PLA. Improvement in
Healthy participants L. plantarum TWK10
Double-blind, Performance assessment: treadmill exercise capacity was dose-dependent. PRO reduced
n = 54, (27/M, 27/F) 3 × 1010 CFU/day or [92]
placebo-controlled running, at 60% VO2max and 85% serum lactate during and after exercise compared to
Age 20–30 y 9 × 1010 CFU/day for 6 weeks
VO2max workload, until exhaustion PLA. Muscle mass increased in the high-dose
PRO group.
L. acidophilus BCMC 12,130, L. PRO did not show superior effects to PLA on
Circuit training protocol, including
Healthy sedentary casei BCMC 12,313, L. lactis BCMC muscular strength (peak torque) and power. PRO
resistance exercises, 3 times a week
individuals Randomized, parallel, 12,451, B. bifidum BCMC 02,290, B. alone and exercise alone increased post-intervention
Performance assessment: muscular [97]
n = 41, males placebo-controlled infantis BCMC 02,129 and B. serum IL-10 concentrations from pre-intervention
strength (peak torque) and power via
Age 19–26 y longum BCMC 02,120 levels. PRO and PLA with or without exercise, had
an isokinetic dynamometer
6 × 1010 CFU/day for 12 weeks no effects on serum IL-6 concentration.
Table 2. Cont.

Intervention
Moderate resistance exercise training,
in instructed classes and at home B. longum BB536, B. infantis M-63,
Healthy elderly individuals An increase in the general cognitive function scores
Randomized, Cognitive assessment: General B. breve M-16V and B. breve B-3
with stretching experience was observed in PRO and PLA groups, at 12 weeks.
double-blind, cognitive performance (incl. tests for 5 × 1010 CFU/day (1.25 × 1010 [112]
Nutrients 2020, 12, 2936
n = 29 (14/M, 25/F) PRO group showed a decrease in anxiety-depression

placebo-controlled accuracy, reaction time), mental state CFU each probiotic/day) for
Age > 65 y scores, body weight, BMI and body fat.
(scoring for depression, anxiety, 12 weeks
and overall mental state)
ICR mice, Institute of Cancer Research mice; L., Lactobacillus (or related genera); B., Bifidobacterium; S., Streptococcus; CFU, colony-forming units; PRO, probiotic supplementation;
PLA, placebo supplementation; CK, creatine kinase; VO2max , maximum rate of oxygen consumption; M, males, F, females; IFN-γ, interferon γ; IL, interleukin; GI, gastrointestinal;
TNF-α = tumor necrosis factor α; ox-LDL, oxidized low-density lipoprotein; MPO, myeloperoxidase; 1RM, 1 repetition maximum; MMP2/9, matrix metalloproteinase 2/9; CRP, C-reactive
protein; HDL, high-density lipoprotein; BMI, body mass index.
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3.3. Improvement in Post-Exercise Recovery

Recovery from exercise represents an important determinant of performance enhancement that
enables adaptation to training. Strategies for optimizing recovery may prevent under-recovery,
overtraining syndrome, injuries, or illnesses [86]. Exercise-induced muscle damage, inflammation,
metabolic responses, and fatigue are part of the recovery process and are, therefore, important
contributors to training adaptation. In addition to physical performance outcomes, biochemical
markers and the athlete’s subjective perception of fatigue and readiness to perform can be assessed,
to evaluate the subject’s recovery state after exercise.
The impacts of probiotic supplementation on health outcomes, performance measurements, and/or
biochemical markers in athletes have been addressed in numerous studies, comparing post-intervention
and pre-intervention resting levels after a training period. Endpoints and markers that are measured
directly after acute exercise sessions and during recovery periods provide a more defined approach to
the evaluation of post-exercise recovery status. The effects of probiotics on biochemical and immune
markers during the post-exercise recovery state after an exercise session have been reported in several
studies (Table 2). The effects of probiotic supplementation on performance capacity during the recovery
period after exercise were studied in triathletes by Huang et al. [105], who assessed anaerobic (Wingate
test) and aerobic (85% VO2max test) exercise capacities, 48 and 72 h after a triathlon race. Probiotic
supplementation (L. plantarum PS128) for 3 weeks significantly improved maximal power, the fatigue
index, and endurance indices during the recovery period after the triathlon race compared with those in
the placebo group. The probiotic group also maintained aerobic performance, when measured during
the recovery period at the resting level, whereas the placebo group reached exhaustion significantly
sooner during the recovery period
High-intensity training acutely increases muscle damage, fatigue, and soreness, which contributed
to decreased athletic performance. Excess mechanical load creates micro-damage to skeletal muscle
tissues, causing local inflammation and decreasing muscle function. Inflammation that occurs in
the muscle tissue is a mechanism of muscular adaptation to exercise, through which the muscle can
regenerate and repair itself [113]. Mechanical overload has been associated with increased systemic
levels of muscle-derived proteins, such as creatine kinase (CK) and myoglobin [107]. Interleukin (IL)-6
is a cytokine that is produced by contracting muscles during exercise and increases in the plasma after
strenuous exercise. Changes in muscle-damage-related biomarkers are associated with delayed-onset
muscle soreness (DOMS) and muscle recovery [114].
In athletes who participated in a full triathlon championship competition, the L. plantarum
PS128 probiotic group and the placebo group did not differ in blood CK values immediately after
competition [105]. However, in the probiotic group, the CK level was significantly lower 3 h
post-exercise compared with that in the placebo group. No differences in post-exercise lactate
dehydrogenase, myoglobin, or free fatty acids were observed between the probiotic and placebo
groups. After less-demanding sprint triathlon, supplementation with L. plantarum PS128 had no effects
compared with placebo on post-exercise CK or blood lactate measurements [105]. In sedentary subjects
who participated in exhaustive exercise, L. plantarum TWK10 improved blood lactate clearance during a
1-h post-exercise recovery period [92]. Blood lactate and lactate clearance are often measured to assess
recovery. However, the suitability of these variables to evaluate fatigue and recovery is controversial
and not agreed on [86].
In the triathlete study performed by Huang et al. [105], the levels of exercise-induced serum
pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, and IL-8,
were significantly lower in the probiotic group compared with those in the placebo group,
both immediately and 3 h after the triathlon competition. The investigators also found increased
anti-inflammatory IL-10 levels after the exercise, but not at the 3-h time point. Prolonged, high-intensity
exercise is well-known to be associated with transient inflammation, immune dysfunction, and oxidative
stress [78]. Lamprecht et al. [72] and Mazani et al. [108] both demonstrated a trend towards reduced
circulating TNF-α levels in the probiotic group compared with the placebo, immediately after exhaustive
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exercise, supporting the findings reported by Huang et al. [105]. Probiotic interventions were found to
increase antioxidant capacity [108], reduce oxidated molecules [72], and decrease myeloperoxidase and
increase thioredoxin activity [105], suggesting overall benefits associated with reduced exercise-induced
oxidative stress levels. However, some probiotic intervention studies have not found any effects on
inflammation [93,100,101,104]; thus, further investigations are warranted to understand the effects of
probiotics on post-exercise immune function and inflammation.
Increased levels of inflammatory cytokines may result from damaged muscle tissue but may
also be caused by the disruption of intestinal barrier function after prolonged, intense, endurance
exercise. Reduced intestinal blood flow causes the acute disruption of epithelial barrier function and
increased leakage, resulting in endotoxemia, during which microbial lipopolysaccharides enter the
blood circulation. The resulting systemic inflammation compromises the athlete’s ability to recover
and perform. Lamprecht et al. [72] showed that a 14-week, multi-strain, probiotic supplementation
protocol reduced fecal zonulin and TNF-α levels significantly compared with those supplemented
with placebo, indicating improved intestinal barrier integrity and reduced systemic inflammation,
respectively. Probiotic supplementation of shorter duration for 4 weeks resulted in reduced
gastrointestinal permeability and improved exercise capacity under heat conditions, with no impacts
on circulating cytokine levels [77]. Moreover, probiotic supplementation did not attenuate exertional
heat stress-induced blood endotoxemia or inflammation [100], the salivary antimicrobial protein
response [101], or extracellular heat shock protein 72 (eHsp72) concentrations [94], when monitored
during the recovery stage, post-exercise. Under normal ambient conditions, a 30-day supplementation
protocol using a multi-strain probiotic did not demonstrate differences in the salivary antimicrobial
peptides during post-exercise recovery after 2 h of cycling at 60% of VO2max [109].
The benefits of probiotic use during recovery from muscle-damaging exercise have been
demonstrated in two clinical studies [106,107]. A study performed in resistance-trained men,
demonstrated that a 3-week supplementation with S. thermophilus FP4 and B. breve BR03 moderately
attenuated post-exercise decreases in muscle performance, as assessed by isometric average peak torque,
24 to 72 h after a muscle-damaging exercise [106]. In addition, circulating IL-6 concentrations were
reduced after the 3-week probiotic supplementation protocol but were not affected by the treatment
during the post-exercise recovery period. Beneficial effects were observed in the resting arm angle after
probiotic supplementation, whereas no differences in flexed arm angle, CK levels, or muscle soreness
were observed during the recovery period, between the probiotic and placebo groups.
A 2-week supplementation of casein combined with Bacillus coagulans BC30, increased perceived
recovery status scores at 24 and 72 h after muscle-damaging exercise compared with casein
supplementation alone in recreationally trained men [107]. Probiotic combined with casein also reduced
perceived muscle soreness compared with casein alone, 72 h post-exercise. Trends toward reduced
circulating CK levels and improved performance, as measured by the Wingate test, were observed
after the muscle-damaging exercise following probiotics combined with casein supplementation
compared with casein supplementation alone. The amounts of muscle swelling and blood urea
nitrogen levels did not differ between the groups. The effects of B. coagulans BC30 have also been
studied among soldiers, who are known to train intensively, on a daily basis, with limited time to recover.
β-Hydroxy-β-methylbutyrate calcium (CaHMB) combined with BC30 maintained muscle integrity
during an intensive 40-day military training period better than CaHMB alone [115]. Treatment with
both CaHMB combined with BC30 and CaHMB alone significantly attenuated resting serum IL-1β,
IL-2, and TNF-α concentrations after the 40-day supplementation period, whereas CaHMB that
was combined with BC30 significantly reduced serum IL-6 and IL-10 during the post-intervention
period compared with control. However, the acute effects on biochemical marker levels during
the recovery state were not evaluated. Probiotics have been proposed to enhance recovery and to
shorten the time necessary for muscle repair by improving the absorption and utilization of dietary
nutrients [107,115,116].
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To date, studies that have assessed performance and exercise capacity during the post-exercise
recovery period remain low in number. Studies that have investigated the probiotic effects on
biochemical and immune markers during the post-exercise recovery period have shown somewhat
controversial results, due to large variations in study designs, training protocols, analytical methods,
athletic populations, and investigated probiotic strains. These results also warrant longer follow-up
measurements during the recovery period. Thus, conclusions cannot be drawn regarding probiotics’
potential to improve recovery and attenuate exercise-induced physiological responses, which are, in part,
necessary for training adaptations and performance enhancement. Furthermore, the relationships
between physiological recovery processes and improvements in performance should be established
more clearly before further conclusions can be made regarding the ergogenic potential of probiotics.
3.4. Improvements in Mood-Related Outcomes

Good physical condition, accompanied by good mental condition, are part of a continuum that
enables the optimal training and performance of competitive athletes. Fatigue and mood disturbances
during performance are common among athletes during the training season and in competition [4].
Intensive exercise causes both physical and psychological stress responses, which can often be difficult
to differentiate between.
Results from preclinical and clinical studies suggest that probiotic administration may have
positive effects in mental responses [117,118]. Few studies have investigated the effects of probiotic
supplementation on the cognitive outcomes of athletes or physically active subjects [84,93,112] (Table 2).
In a group of highly trained, elite athletes, the self-rated sense of vigor (Profile of Mood States,
POMS questionnaire) was significantly increased among the probiotic group, who ingested L. helveticus
Lafti for 14 weeks, compared with the placebo group, with no difference in the total mood disturbance
scores between groups detected [84]. Decreased vigor is related to an individual’s feelings of possessing
the necessary physical strength to perform.
In a study involving highly trained, female, competitive swimmers, probiotic supplementation
(B. longum 35,624) during a 6-week intensive training period improved the cognitive functions of the
athletes [93]. At the end of the intensive training period, significant differences in the scores related
to sport recovery categories (the Recovery-Stress Questionnaire for Athletes) were detected between
groups, showing that the scores of the probiotic group were more favorable compared with those
in the placebo group. A training intervention performed in healthy, elderly, Japanese individuals
demonstrated that a 12-week resistance training program induced beneficial effects on the general
cognitive functions of both the placebo and probiotic groups [112]. The 12-week supplementation with
multi-strain bifidobacteria significantly decreased overall mental state scores compared with baseline
scores, with lower scores indicating lower depression and anxiety symptoms [112].
Probiotics are, by definition, live microorganisms. In addition to viable bacteria, studies have been
performed using inactivated bacteria. Two studies have investigated the effects of supplementation with
inactivated Lactobacillus on mood related measurements [119,120]. A 4-week supplementation period,
using heat-inactivated L. gasseri OLL2809, reduced tension-anxiety scores after a 1-h cycle ergometer
exercise, compared with baseline scores [119]. The 12-week administration of heat-inactivated L. gasseri
CP2305 significantly decreased scores that measured physical fatigue, anxiety, and depression in male
university student-athletes [120]. Salivary cortisol and chromogranin A serve as biochemical markers
for stress. In the above-mentioned studies, salivary chromogranin A was significantly reduced in the
inactivated Lactobacillus group compared with that in the placebo group [120], whereas no changes in
salivary cortisol levels were detected after the intervention period [119,120].
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Probiotics appear to have benefits on cognitive outcomes in athletes, as measured by self-reported

scores. Several potential mechanisms exist for the gut bacteria to interact with the brain, through
the gut-brain axis. Messages to the brain can be delivered by gut-derived cytokines, hormones,
and bacterial metabolites, including neurotransmitters, or via the vagus nerve [4]. Probiotic studies
that focus on the mental health of athletes represent an emerging area in the field of sports nutrition
and exercise performance. The number of probiotic studies remains very low, with studies often
including a low number of subjects, and a wide variety of questionnaires have been used to assess
cognitive outcomes. Despite limited evidence, cognitive health remains an intriguing area of sports
nutrition research.
4. Conclusions
Overall, growing evidence from animal and human studies has indicated that the gut microbiota
composition plays an important role in host physiology and can affect physical performance.
The microbial community of the gut and its potential health benefits are highly impacted by
individual life choices, including dietary patterns and activity levels. Probiotics are known for
their potential to reduce GI and URT symptoms and infection episodes and thus may benefit the athlete
by increasing the numbers of healthy training days and completed races. Further, probiotics may
support athletic performance by enhancing training adaptations, attenuating physiological responses
during post-exercise recovery periods, and improving mood and mental responses after intense exercise.
Therefore, probiotics can be considered to act as indirect ergogenic aids; however, the causal impacts of
indirect effects on performance remain to be established in good-quality, long-term studies of adequate
size that consider the diet, and the training and competition seasons of the athletes. The functions of
probiotics in enhancing performance, as direct ergogenic aids, require additional research that targets
the mode of action that underlies their potential benefits.
Author Contributions: Conceptualization, M.M., R.A.-J., A.L., and M.J.L.; writing—original draft preparation,
M.M. and R.A.-J.; writing—review and editing, M.M., R.A.-J., A.L., and M.J.L.; visualization, M.M. and R.A.-J.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: Arthur Ouwehand and Johanna Maukonen are acknowledged for their comments during the
manuscript preparation.
Conflicts of Interest: The authors are employees of Danisco Sweeteners Oy, legal entity of DuPont Nutrition &
Biosciences that manufactures probiotics.
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nutrients
Article
High Salt Diet Impacts the Risk of Sarcopenia
Associated with Reduction of Skeletal Muscle
Performance in the Japanese Population
Yasuko Yoshida 1,2, *, Keisei Kosaki 3,4,5 , Takehito Sugasawa 2 , Masahiro Matsui 5,6 ,
Masaki Yoshioka 6 , Kai Aoki 6 , Tomoaki Kuji 6 , Risuke Mizuno 7 , Makoto Kuro-o 8 ,
Kunihiro Yamagata 9 , Seiji Maeda 4 and Kazuhiro Takekoshi 2
1 Department of Clinical Laboratory Science, Faculty of Health Sciences, Tsukuba International University,
Ibarak 300-0051, Japan
2 Laboratory of Sports Medicine, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba,
Ibaraki 305-8577, Japan; [email protected] (T.S.); [email protected] (K.T.)
3 Faculty of Sport Sciences, Waseda University, Saitama 359-1192, Japan; [email protected]
4 Faculty of Health and Sport Sciences, University of Tsukuba, Ibaraki 305-8577, Japan;
[email protected]
5 Japan Society for the Promotion of Science, Tokyo 102-0083, Japan; [email protected]
6 Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki 305-8577, Japan;
[email protected] (M.Y.); fi[email protected] (K.A.); [email protected] (T.K.)
7 Faculty of Veterinary Medicine, Okayama University of Science, Ehime 794-8555, Japan;
[email protected]
8 Division of Anti-aging Medicine, Center for Molecular Medicine, Jichi Medical University,
Tochigi 329-0431, Japan; [email protected]
9 Department of Nephrology, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8577, Japan;
[email protected]
Received: 16 October 2020; Accepted: 5 November 2020; Published: 12 November 2020
Abstract: The World Health Organization has recommended 5 g/day as dietary reference intakes
for salt. In Japan, the averages for men and women were 11.0 g/day and 9.3 g/day, respectively.
Recently, it was reported that amounts of sodium accumulation in skeletal muscles of older people
were significantly higher than those in younger people. The purpose of this study was to investigate
whether the risk of sarcopenia with decreased muscle mass and strength was related to the amount of
salt intake. In addition, we investigated its involvement with renalase. Four groups based on age
and salt intake (“younger low-salt,” “younger high-salt,” “older low-salt,” and “older high-salt”)
were compared. Stratifying by age category, body fat percentage significantly increased in high-salt
groups in both younger and older people. Handgrip strength/body weight and chair rise tests of the
older high-salt group showed significant reduction compared to the older low-salt group. However,
there was no significant difference in renalase concentrations in plasma. The results suggest that
high-salt intake may lead to fat accumulation and muscle weakness associated with sarcopenia.
Therefore, efforts to reduce salt intake may prevent sarcopenia.
Keywords: salt; sarcopenia; renalase; body fat percentage; knee extensor muscle strength; single-leg
stance time; maximum gait speed; long seat type body anteflexion; chair rise test
1. Introduction
Japan has a large aging population; the 2019 White Paper on Aging Society reported that 28.1% of
the population is over the age of 65 years [1]. In addition, soaring medical costs have become a social
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problem. One of the causes is that there is a discrepancy between the average life expectancy and
the period of healthy life expectancy during which a person can live an independent life [1]. Frailty,
the decline in motor and cognitive function in individuals with advanced age, is one of the reasons
healthy life expectancy remains lower than average life expectancy. The main cause for frailty is
sarcopenia, a progressive age-related weakness of the muscles. The definition of sarcopenia was based
on the criteria of the European Working Group on Sarcopenia in Older People (EWGSOP) in 2010 [2].
In Japan, the criteria of the Asian Working Group for Sarcopenia (AWGS) are generally recommended
for diagnosis, and in 2017 the Japanese Association on Sarcopenia and Frailty created the sarcopenia
clinical practice guidelines [3–5].
The main cause of sarcopenia is aging [2–4,6]. Secondary factors include physical inactivity,
illness, and nutrition, and it is likely that multiple factors overlap to cause sarcopenia. Specifically,
with regard to nutrition, reports indicate that deficiency of protein and vitamin D intake increases the
risk of sarcopenia [7–21]. The leading problem regarding nutrients in Japanese people is high-salt
intake. The World Health Organization (WHO) has recommended an intake of no more than 5 g of
salt/day [22]. In Japan, there is an average salt intake of 11.0 g/day for men and 9.3 g/day for women.
Recently in Japan, salt intake has been declining slightly each year [23].
Many studies report that excessive salt intake is one of the causes of various diseases, such as
hypertension [24,25], where sodium in the skeletal muscle accumulates more in older people than
in younger people, and patients with refractory hypertension have increased tissue sodium(Na (+))
content when compared with normotensive controls [26]. In addition, the sodium-potassium-chloride
symporter 1 (NKCC1) is highly expressed in mammalian skeletal muscle. The physiologic function
of NKCC1 in myogenesis is unclear. However, NKCC1 protein levels increased skeletal myoblast
differentiation, and NKCC1 inhibitors markedly suppressed skeletal myoblast differentiation [27].
It has also been reported that excess sodium leads to a downregulation of expression of NKCC [28].
Therefore, we hypothesized that the risk of sarcopenia, which is associated with decreased muscle
mass and strength, is related to the amount of salt intake, because Na (+) is stored in tissues and
NKCC1 is involved in muscle hypertrophy and suppression.
Incidentally, renalase, a recently discovered enzyme released by the kidneys, may break down
blood-derived catecholamine and regulate blood pressure. A report of loss of renalase function can
result in increased blood pressure (hypertension), increased heart rate (tachycardia), increased vascular
resistance (vasoconstriction), and increased catecholamine response [29]. Human and animal studies
have suggested that high levels of dietary salt may lower blood and renal renalase levels. Due to blood
pressure increasing with salt intake, the renalase levels that breaks down catecholamines, may also be
reduced [30–32]. For the relationship between renalase and skeletal muscle, exercise load increased
blood levels of renalase, independent of renal secretion, and increased gene expression of renalase
in skeletal muscles [33–35]. In a study based on disuse atrophy, there was a study that showed
muscle proteolysis decreased and muscle mass increased in renalase knockout mice [36]. On the other
hand, skeletal muscle has β2 adrenergic receptors involved in catecholamines, and β2-adrenergic
receptor stimulation increases muscle mass by promoting muscle protein synthesis and/or attenuating
protein degradation. However, excessive stimulation of -adrenergic receptors negates their beneficial
effects [37]. By these reports, we considered that renalase, which is related to catecholamines that
increase with salt intake, may also be involved in skeletal muscle atrophy. Therefore, we speculated
that the decreased level of renalase might be a marker for sarcopenia.
We aimed to clarify the relationship between salt intake, blood renalase concentrations,
and sarcopenia risk for Japanese adults.
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2.1. Study Design

Healthy adult volunteers (n = 122, age (standard deviation [SD]): 56.3 (12.3) years) were recruited
from the community using local advertisements, and participants were selected from Tsukuba and
nearby urban areas (Ibaraki, Japan). Participants with an estimated glomerular filtration rate of
90 mL/min/1.73 m2 or less were excluded because renal function is involved in sodium uptake and
renalase levels [24,30–32,38–40]. In addition, eight participants who showed more than four times
the standard deviation of the mean blood test of all 122 participants were excluded, because there
was a possibility of other diseases. The items are renalase, Interleukin-6 (IL-6), triglyceride (TG),
insulin, glucose (Glu), glycosylated hemoglobin (HbA1c), aspartate transaminase (AST), and alanine
aminotransferase (ALT). Therefore, 114 participants were analyzed.
We calculated estimated salt intake using spot urine testing and divided the participants into
two groups with an average estimated salt intake of 9.37 g/day [1]. The participants with lower salt
intake than average was designated as “low-salt,” and the participants with a higher intake were
designated as “high-salt.” Furthermore, since the age range of the selection criteria for this study is
20 years and over, volunteers from a very wide range of age groups participated, we also analyzed
after stratifying by age. Participants were divided according to the overall average age (56.3 (12.3))
designated “younger” and “older.” That is, “younger” is younger than the average age of participants
and “older” is older than the average age. Overall, the participants were divided into four groups
(“younger low-salt,” “younger high-salt,” “older low-salt,” “older high-salt”) and blood test results,
anthropometric measurements, and assessment of physical performance were compared.
This study was approved by the Ethical Review Board of University of Tsukuba, and all participants
provided written, informed consent as per the requirements of the Declaration of Helsinki (IRB approval
No. H30-161).
2.2. Data Collection
2.2.1. Blood Test and Urinalysis

Participants were asked to avoid strenuous exercise, live a normal life the day before and were
banned from eating and drinking except water after 20:00 p.m., before testing to control for exercise
and nutrition. They were asked to arrive at the laboratory between 8:00 a.m. and 11:00 a.m.
First, participants provided urine samples. We conducted urinalysis to measure the concentration
of sodium and creatinine at Tsukuba i-Laboratory Limited Liability Partnership (Ibaraki, Japan), using
the ion-selective electrode method to measure urinary sodium and the enzyme method to measure
urinary creatinine. We calculated the 24-h salt intake using the following formula [41,42]:
Estimated 24 h Creatinine (mg/day) = −2.04 × age + 14.89 × weight (kg) + 16.14 × height (cm) − 2244.45, (1)
Estimated 24 h urinary sodium (mEq/day) = 21.98 × urinary sodium/10/(1) 0.392 , (2)
Estimated 24-h salt intake (g/day) = (2)/17. (3)
Next, blood samples were collected from the antecubital vein. In this study, we measured some
parameters in the blood that could pose a risk for sarcopenia. IL-6 (pg/mL) for inflammation,
urea nitrogen (UN) (mg/dL) and cystatin C (CysC) (mg/L) for renal function, TG (mg/dL) for
fat accumulation that causes obesity, albumin (Alb) (g/dL) for nutritional status, Glu (mg/dL),
insulin (μU/mL) and HbA1c (%), for glucose metabolism, and AST (U/L) and ALT (U/L) for liver function.
In addition, since renalase (mg/L) has been reported to be involved in blood pressure control function
and skeletal muscle atrophy by metabolizing catecholamines, it was measured as a marker candidate
for sarcopenia. Whole blood was used to measure HbA1c, which was measured using the enzyme
method. The remaining blood was brought to room temperature of 20 to 25 ◦ C and then centrifuged at
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3000 rpm for 10 min to obtain a serum. It was then stored at −80 ◦ C until measurement. Serum was
used for measurement of concentrations of the following parameters: IL-6, UN, CysC, TG, Alb, insulin,
Glu, AST, ALT, and renalase. The measurement was carried out at Tsukuba i-Laboratory Limited
Liability Partnership (Ibaraki, Japan) by chemiluminescent enzyme immunoassay (insulin), ultraviolet
(UN, Glu), latex coagulating nephelometry (CysC), the enzyme method (TG), the bromocresol purple
method (Alb), and the Japan Society of Clinical Chemistry (JSCC) Standardization Corresponding
Method (AST, ALT). IL-6 was measured by Chemiluminescent Enzyme Immunoassay at Jichi Medical
University Hospital. Renalase was measured by enzyme-linked immunosorbent assay (ELISA) using
the FAD-Dependent Amine Oxidase ELISA Kit (Cloud-Clone Corp, Houston, TX, USA). In addition,
the estimated glomerular filtration value (eGFR) was calculated using the renal function presumption
formula and serum CysC concentrations to evaluate kidney function. Moreover, eGFR using the
serum CysC concentration was calculated using the equation for Japanese people as shown in the
chronic kidney disease (CKD) clinical practice guidelines [40] as follows: eGFR (mL/min/1.73 m2 ) =
(104 × [Concentration of serum CysC (mg/dL)]−1.019 × 0.996(Age) − 8”.
2.2.2. Anthropometric Measurements

Heights were measured using a stadiometer. Weight and body compositions were then measured
using bioelectrical impedance analysis (Inbody 770; Inbody, Tokyo, Japan). The body composition
measurement included skeletal muscle mass index (SMI; kg/m2 ), body fat percentage (BFP; %), and mass
of muscle of upper limbs (ULMM; kg) and lower limbs (LLMM; kg) [43,44]. Then we calculated the
body mass index (BMI; kg/m2 )
2.2.3. Assessment of Physical Performance

Handgrip strength (HGS) (kg), knee extensor muscle strength (KES) (kg), single-leg stance time
(SLT) (s), maximum gait speed (MGS) (m/s), long seat type body anteflexion (FleX) (cm), and the
chair rise test (30CS) (number of repetitions) were used as indices of physical performance [3–5,45–51].
HGS was determined using a handheld dynamometer (T.K.K.5401; Takei Kiki Kogyo, Niigata, Japan).
The participants being tested alternated between their left and right hand and were measured twice.
The mean of the maximal values for each hand was used for analysis [49,50]. Then, the HGS was
divided by body weight (BW). KES was determined using a handheld dynamometer (μTas F-1; ANIMA,
Tokyo, Japan). Both lower limbs were measured twice, and the average of their highest score was
registered as the result. Then, the KES was divided by body weight [48,50]. SLT was measured as an
indicator of balance performance. Participants balanced as long as possible on one leg with their eyes
open. The participant could choose which foot on which to balance. The maximum value was 60 s.
If the maximum value was achieved, it was completed once; if not, the test was carried out twice and
the longest time was used [47,50]. MGS was calculated using the time to cover a distance of 10 m
on a straight walking course. To ensure that the participants reached their maximal walking speed,
the 10 m was measured within a 16-m track [46,50]. FleX was determined using the long seat type body
anteflexion measurement device (T.K.K.5412; Takei Kiki Kogyo, Niigata, Japan) [45]. The measurement
was performed twice, and the largest value was used as the data. Muscular function of lower extremities
was measured by 30CS as described by Jones et al. [51]. Participants were instructed to stand up as
often as possible within 30 s. The score was the total number of stands executed correctly (stretched
knee and hip) within 30 s. The measurement was performed only once.

Data were analyzed using SPSS Statistics 26.0 (IBM, Armonk, IL, USA). Significance was set at
p < 0.05. All values are expressed as mean (standard deviation) or median [interquartile range] unless
stated otherwise. A chi-square test was performed to confirm that there was no difference in the ratio of
males and females in each group. Normal distribution was confirmed by a Kolmogorov–Smirnov test.
In the comparison between two groups, an independent t-test was performed in the case of normal
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distribution, and the Mann–Whitney U test was performed in other cases. For unequal variance,
Welch’s correction was applied. The correlation was analyzed by Pearson’s correlation coefficient and
Spearman’s correlation coefficient. In the comparison between the four groups, one-way analysis of
variance (ANOVA) for normality was performed, and Kruskal–Wallis for non - normality testing was
performed otherwise. If there was a significant difference, either the Tukey test or the Bonferroni test
was applied as a post hoc test. For multiple analysis, multiple linear regression analysis was performed
with salt intake as the dependent variable.
3. Results
3.1. Comparison with Estimated Daily Salt Intake

The normality of each parameter is shown in Table 1. All parameters except BFP, HGS/BW,
KES/BW, Flex, UN, Alb, HbA1c, and AST showed a non-normal distribution. The comparison between
the “low-salt” and “high-salt” groups is shown in Table 1. There were no significant differences in
sex ratio, age, and height. However, there were significant differences in SBP, DBP, weight, BMI, BFP,
HGS/BW, Flex, 30CS, IL-6, TG, insulin, and ALT.
Table 1. Comparison of sarcopenia-related parameters by estimated salt intake.
Normality T Test
Low—Salt High—Salt
p Value
p Value
Sample size (n) 57 57 - -
Salt intake (g/day) 7.62(1.31) 11.11(1.16) 0.63 <0.01 *
Male/Female (n/n) 11/46 8/49 - 0.45 †
Age (year) § 56.00[49.00–63.00] 56.00[50.5–65.00] <0.01 * 0.60
SBP (mmHg) § 113.00[104.33–122.00] 117.33[110.33–128.00] <0.01 * 0.05 *
DBP (mmHg) § 65.67[60.00–73.00] 70.33[66.17–76.00] 0.04 * 0.01 *
Height (m) § 1.59 [1.53–1.66] 1.57[1.52–1.61] <0.01 * 0.28
Weight (kg) § 51.20[45.30–58.30] 57.60[48.75–66.25] <0.01 * 0.01 *
BMI (kg/m2 ) § 20.61[18.88–21.89] 22.69[20.28–25.58] <0.01 * <0.01 *
SMI (kg/m2 ) § 6.10[5.50–6.90] 6.20[5.80–6.85] <0.01 * 0.34
ULMM (kg) § 3.56[2.95–4.32] 3.67[3.24–4.35] <0.01 * 0.24
LLMM (kg) § 11.69[10.04–14.19] 11.78[10.44–12.82] <0.01 * 0.88
BFP (%) 23.21(7.15) 29.91(8.65) 0.10 <0.01 *
HGS/BW (kg/BW) 0.54(0.10) 0.46(0.12) 0.23 <0.01 *
KES/BW (kg/BW) 0.62(0.17) 0.57(0.16) 0.16 0.13
SLT (sec) § 60.00[48.05–60.00] 60.00[38.24–60.00] <0.01 * 0.72
MGS (m/sec) § 2.28[2.04–2.61] 2.18[1.99–2.44] <0.01 * 0.14
Flex (m) 0.39(9.08) 0.36(7.54) 0.75 0.04 *
30CS (time) § 24.00[19.00–29.00] 18.00[16.00–23.00] <0.01 * <0.01 *
Renalase (mg/L) § 4.26[2.93–5.46] 4.24[3.30–5.38] <0.01 * 0.90
IL-6 (pg/mL) § 1.00[0.65–1.20] 1.20[0.90–1.90] <0.01 * <0.01 *
UN (mg/dL) 14.04(2.81) 13.07(2.61) 0.10 0.06
CysC (mg/L) § 0.65[0.61–0.69] 0.67[0.62–0.70] <0.01 * 0.27
TG (mg/dL) § 61.00[46.00–78.00] 83.00[59.50–100.50] <0.01 * <0.01 *
Alb (g/dL) 4.53(0.30) 4.51(0.26) 0.10 0.77
Glu (mg/dL) § 96.00[91.00–106.00] 99.00[92.50–104.00] <0.01 * 0.28
Insulin (μU/mL) § 3.90[2.90–6.10] 4.90[3.75–7.15] <0.01 * 0.02 *
HbA1c (%) 5.61(0.33) 5.69(0.38) 0.11 0.27
AST (U/L) 22.93(4.93) 24.11(5.03) 0.70 0.21
ALT (U/L) § 17.00[14.00–19.00] 18.00[15.00–27.00] <0.01 * 0.05 *
These data are shown as mean (standard deviation) or median [interquartile range]. For the normality test,
the Kolmogorov—Smirnov test was performed. Two-group comparison is the result of the independent T test or
Mann—Whitney-U test [§]. The statistics of male and female is the chi-square test [†]. *: p < 0.05. sec: seconds,
BW: body weight, SBP: systolic blood pressure, DBP: diastolic blood pressure, BMI: body mass index, SMI: skeletal
muscle mass index, ULMM: mass of muscle of upper limbs, LLMM: mass of muscle of lower limbs, BFP: body fat
percentage, HGS/BW: handgrip strength/weight, KES: knee extensor muscle strength, SLT: single-leg stance time,
MGS: maximum gait speed, Flex: long seat type body anteflexion, 30CS: chair rise test, IL-6: Interleukin-6, UN: urea
nitrogen, CysC: cystatin C, TG: triglyceride, Alb: albumin, Glu: glucose, HbA1c: Glycosylated hemoglobin, AST:
Aspartate transaminase, ALT: Alanine aminotransferase.
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3.2. Correlation with Estimated Salt Intake

The correlations between estimated salt intake and sarcopenia-related parameters are shown in
Table 2. As for the correlation coefficient, the correlation coefficient of Pearson was analyzed in the case
of normality, and the correlation coefficient of spearman was analyzed in the case of non-normality.
There were significant differences in SBP, Weight, BMI, SMI, ULMM, BFP, HGS/BW, Flex, 30Cs, IL-6,
TG, Insulin, and ALT. SBP, Weight, SMI, ULMM, HGS/BW, Flex, 30CS, IL-6, TG, Insulin, and ALT was
weakly correlated and BMI and BFP was moderately correlated.
Table 2. Correlation of sarcopenia-related parameters based on estimated salt intake.
Correlation Coefficient
p Value r Value
Age (year) § 0.54 -
SBP (mmHg) § 0.03 * 0.21
DBP (mmHg) § 0.07 -
Height (m) § 0.74 -
Weight (kg) § <0.01 * 0.39
BMI (kg/m2 ) § <0.01 * 0.49
SMI (kg/m2 ) § 0.03 * 0.21
ULMM (kg) § 0.01 * 0.24
LLMM (kg) § 0.42
BFP (%) <0.01 * 0.49
HGS/BW (kg/BW) <0.01 * −0.38
KES/BW (kg/BW) 0.14 −
SLT (sec)§ 0.46 −
MGS (m/sec) § 0.24 −
Flex (m) 0.03 * −0.20
30CS (time) § <0.01 * −0.32
Renalase (mg/L) § 0.67 −
IL-6 (pg/mL) § <0.01 * 0.31
UN (mg/dL) 0.25 −
CysC (mg/L) § 0.15 −
TG (mg/dL) § <0.01 0.34
Alb (g/dL) 0.67 −
Glu (mg/dL) § 0.11 −
Insulin (μU/mL) § <0.01 * 0.27
HbA1c (%) 0.12 −
AST (U/L) 0.51 −
ALT (U/L) § 0.02 * 0.21
This table shows the correlation between estimated salt intake and sarcopenia-related parameters. As for the
correlation coefficient, the Pearson correlation coefficient was shown in the case of normality, and the Spearman
correlation coefficient was shown in the case of non-normality [§]. *: p < 0.05. sec: seconds, BW: body weight, SBP:
systolic blood pressure, DBP: diastolic blood pressure, BMI: body mass index, SMI: skeletal muscle mass index,
ULMM: mass of muscle of upper limbs, LLMM: mass of muscle of lower limbs, BFP: body fat percentage, HGS/BW:
handgrip strength/weight, KES: knee extensor muscle strength, SLT: single-leg stance time, MGS: maximum gait
speed, Flex: long seat type body anteflexion, 30CS: chair rise test, IL-6: Interleukin-6, UN: urea nitrogen, CysC:
cystatin C, TG: triglyceride, Alb: albumin, Glu: glucose, HbA1c: Glycosylated hemoglobin, AST: Aspartate
transaminase, ALT: Alanine aminotransferase.
3.3. Comparison of Estimated Salt Intake and Age

The comparison between the “younger low-salt,” “younger high-salt,” “older low-salt,” and
“older high-salt” groups is shown in Table 3. There were significant differences in SBP, DBP, weight,
BMI, SMI, ULMM, LLMM, BFP, HGS/BW, 30CS, IL-6, UN, TG, Glu, HbA1c, and AST.
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Table 3. Comparison of sarcopenia-related parameters by estimated salt intake and age.
Younger Low—Salt Younger High—Salt Older Low—Salt Older High—Salt p Value

Sample size (n) 26 25 31 32 –
Salt intake (g/day) 7.56(1.19) 11.01(1.08) 7.67(1.42) 11.19(1.23) –
Male/Female (n) 5/21 6/19 6/25 2/30 –
Age (year) § 48.50[37.75–53.00] 50.00[44.50–54.00] 62.00[58.00–71.00] 65.00[60.25–67.00] –
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SBP (mmHg) § 115.33[103.33–122.67] 112.67[103.83–119.83] 112.33[105.75–121.00] 122.50[113.67–130.75] <0.01 *

DBP (mmHg) § 68.17[60.50–73.75] 68.33[64.17–72.50] 65.33[59.67–71.67] 72.67[67.83–81.50] <0.01 *
Weight (kg) § 52.50[46.23–62.33] 61.50[52.80–69.00] 47.00[44.80–56.80] 51.15[47.83–62.13] <0.01 *
BMI (kg/m2 ) § 20.61[19.00–22.50] 23.10[20.50–25.86] 20.45[18.27–21.83] 22.20[20.00–25.61] <0.01 *
SMI (kg/m2 ) § 6.25[5.78–7.10] 6.40[6.05–7.60] 6.00[5.40–6.70] 5.95[5.63–6.78] 0.01 *
ULMM (kg) § 3.81[3.19–4.71] 3.82[3.56–5.55] 3.28[2.86–4.04] 3.61[2.90–4.13] 0.03 *
LLMM (kg) § 12.44[10.21–14.43] 12.27[11.67–17.10] 11.04[9.92–13.63] 10.86[9.91–12.15] <0.01 *
BFP (%) 22.54(7.31) 28.38(7.98) 23.76(7.08) 31.12(9.07) <0.01 *
HGS/BW (kg/BW) 0.56(0.10) 0.49(0.11) 0.52(0.10) 0.44(0.13) <0.01 *
KES/BW (kg/BW) 0.64(0.20) 0.62(0.16) 0.60(0.14) 0.53(0.16) 0.08
SLT (sec) § 60.00[60.00–60.00] 60.00[48.35–60.00] 60.00[25.12–60.00] 60.00[37.51–60.00] 0.23
MGS (m/sec) § 2.34[2.13–2.79] 2.25[2.05–2.73] 2.24[1.95–2.59] 2.13[1.81–2.33] 0.05
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Flex (m) 0.39(0.10) 0.36(0.09) 0.38(0.11) 0.35(0.07) 0.44
30CS (time) § 24.00[19.75–29.00] 20.00[16.00–23.00] 21.00[18.00–29.00] 17.50[16.00–22.00] <0.01 *
Renalase (mg/L) § 3.71[2.78–6.30] 4.85[2.71–6.28] 4.40[3.37–5.12] 4.13[3.45–4.95] 0.73
IL-6 (pg/mL) § 0.80[0.60–1.03] 0.90[0.75–1.45] 1.00[0.70–1.30] 1.70[1.10–2.08] <0.01 *
UN (mg/dL) 13.23(2.87) 12.54(2.29) 14.72(2.62) 13.49(2.79) 0.02 *
CysC (mg/L) § 0.63[0.61–0.68] 0.66[0.61–0.69] 0.65[0.62–0.70] 0.68[0.61–0.70] 0.43
TG (mg/dL) § 65.50[46.50–81.75] 84.00[52.50–95.00] 58.00[43.00–78.00] 82.50[66.25–105.75] 0.01 *
Alb (g/dL) 4.50(0.29) 4.54(0.24) 4.55(0.32) 4.49(0.27) 0.74
Glu (mg/dL) § 93.00[87.50–99.25] 97.00[90.50–100.50] 99.00[94.00–108.00] 100.00[94.25–107.75] 0.02 *
insulin (μU/mL) § 4.05[2.98–5.95] 4.90[3.55–6.55] 3.70[2.70–6.80] 5.00[3.98–7.45] 0.10
HbA1c (%) 5.48(0.29) 5.55(0.31) 5.73(0.32) 5.79(0.39) <0.01 *
AST (U/L) 21.19(4.50) 23.36(5.06) 24.39(4.87) 24.69(5.01) 0.04 *
ALT (U/L) § 16.00[12.00–17.25] 19.00[15.00–29.50] 18.00[15.00–21.00] 16.00[15.00–26.75] 0.05
These data are shown as mean (standard deviation) or median [interquartile range]. This is the result of one-way ANOVA or Kruskal-Wallis tests [§]. *: p < 0.05. sec: seconds, BW: body
weight, SBP: systolic blood pressure, DBP: diastolic blood pressure, BMI: body mass index, SMI: skeletal muscle mass index, ULMM: mass of muscle of upper limbs, LLMM: mass of
muscle of lower limbs, BFP: body fat percentage, HGS/BW: handgrip strength/weight, KES: knee extensor muscle strength, SLT: single-leg stance time, MGS: maximum gait speed, Flex:
long seat type body anteflexion, 30CS: chair rise test, IL-6: Interleukin-6, UN: urea nitrogen, CysC: cystatin C, TG: triglyceride, Alb: albumin, Glu: glucose, HbA1c: Glycosylated
hemoglobin, AST: Aspartate transaminase, ALT: Alanine aminotransferase.
Nutrients 2020, 12, 3474
In the comparison between the four groups, one-way analysis of variance (ANOVA) for normality
was performed while Kruskal-Wallis was performed for non-normality testing. Significant differences
were found in SBP, DBP, Weight, BMI, SMI, ULMM, LLMM, BFP, HGS/BW, 30CS, IL-6, UN, TG, Glu,
HbA1c, and AST. A post hoc test was calculated only on the parameters that were significantly different.
Figure 1 shows the p-values of the parameters that were significantly different in the post hoc test.
When Younger and Older are stratified, significant differences were found in SPB, DBP, BMI, HGS/BW,
30CS, IL-6, and TG only in the older group. BFP was significantly different in both younger and older
groups. weight, SMI, ULMM, LLMM, UN, Glu, HbA1c, and AST were significantly different between
the younger group and the older group.
Figure 1. Cont.
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IL-6 UN CysC
6 <0.01* 25 0.02* 1.0
0.03*
20 0.8
4
(pg/mL)
(mg/dL)
(mg/L)
15 0.6
10 0.4
2
5 0.2
0 0 0.0
t
t
lt
lt
lt
lt
lt
lt
al
al
al
al
al
al
sa
sa
sa
sa
sa
sa
-s
-s
-s
-s
-s
-s
h-
h-
h-
h-
h-
h-
w
w
ig
ig
ig
ig
ig
ig
Lo
Lo
Lo
Lo
Lo
Lo
H
H
TG Alb Glu
300 6.0 140 0.03*
0.02*
5.5
120
200
(mg/dL)
(mg/dL)
(g/dL) 5.0
100
4.5
100
80
4.0
0 3.5 60
t
t
t
t
t
t
al
al
al
al
al
al
al
al
al
al
al
al
-s
-s
-s
-s
-s
-s
-s
-s
s
s
h-
h-
h-
h-
w
w
h
w
ig
ig
ig
ig
ig
ig
Lo
Lo
Lo
Lo
Lo
Lo
H
H
Insulin HbA1c AST
<0.01* 0.04*
25 7.0 40
0.04*
0.03*
20 6.5
30
(ΐ U /m L)
15 6.0
(U/L)
(%)
20
10 5.5
10
5 5.0
0 4.5 0
t
t
lt
lt
lt
lt
lt
lt
al
al
al
al
al
al
sa
sa
sa
sa
sa
sa
-s
-s
-s
-s
-s
-s
h-
h-
h-
h-
h-
h-
w
w
ig
ig
ig
ig
ig
ig
Lo
Lo
Lo
Lo
Lo
Lo
H
H
ALT
80
60
(U/L)
40
20
0
t
t
lt
lt
al
al
sa
sa
-s
-s
h-
h-
w
w
ig
ig
Lo
Lo
H

Figure 1. For comparison of the four groups, one-way ANOVA or Kruskal-Wallis test was performed,
followed by a post hoc test. *: p < 0.05. sec: seconds, BW: body weight, SBP: systolic blood pressure,
DBP: diastolic blood pressure, BMI: body mass index, SMI: skeletal muscle mass index, ULMM: mass
of muscle of upper limbs, LLMM: mass of muscle of lower limbs, BFP: body fat percentage, HGS/BW:
handgrip strength/weight, KES: knee extensor muscle strength, SLT: single-leg stance time, MGS:
maximum gait speed, Flex: long seat type body anteflexion, 30CS: chair rise test, IL-6: interleukin-6,
UN: urea nitrogen, CysC: cystatin C, TG: triglyceride, Alb: albumin, Glu: glucose, HbA1c: glycosylated
hemoglobin, AST: aspartate transaminase, ALT: alanine aminotransferase.
3.4. Multivariate Analysis of Estimated Salt Intake and Sarcopenia Parameters

Multiple linear regression analysis used salt intake as the dependent variable. The independent
variable populated all parameters related to sarcopenia risk. We then implemented the stepwise method.
The adopted factors were BFP, ULMM, IL-6, SLT, LLMM, 30CS, ALT, and insulin, and the multiple
regression equation for predicting salt intake (Y) was Y = 0.12 × (BFP) + 1.44 × (ULMM) + 0.46 × (IL-6)
+ 0.03 × (SLT) − 0.42 × (LLMM) − 0.08 × (30CS) + 0.05 × (ALT) − 0.14 × (insulin) + 5.09. The multiple
correlation coefficient was 0.46, the adjusted coefficient of determination was 0.42, and the adjusted
multiple correlation coefficient was 0.68. The value of the Burbin Watson test was 2.40. Residual
analysis showed that the model was reliable.
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Nutrients 2020, 12, 3474
4. Discussion
This study was conducted with a focus on salt intake, which pertains to nutritional intake.
It is known that the salt intake of Japanese people is the highest in the world [52,53]. In this study,
the estimated daily salt intake using the urine test values was high, with an average value of 9.32 g/day
(2.14). This amount is close to the reported average Japanese salt intake and is almost twice the
recommended amount given by the WHO [22,23].
All parameters that showed a significant difference are values indicating the degree of obesity
and the amount of fat in the body. In addition, salt intake, BFP, and BMI showed positive correlations.
These results suggest that excessive salt intake may contribute to fat accumulation and are consistent
with previous studies reporting an association between excessive salt intake and obesity [18,54–56].
One factor that predicts sarcopenia is the increase of fat in muscle [57–60]. Excessive salt intake can
lead to fat accumulation and sarcopenia risk, however, participants who consume excess salt may
not have dietary controls other than salt, which may lead to fat accumulation and obesity. Since no
dietary survey was conducted in this study, this cannot be clarified. In terms of assessment of physical
performance, some parameters, such as muscle weakness, were significantly lower in the high-salt
group than the low-salt group. Muscle weakness is one of the causes of sarcopenia [50,61]. However,
according to the criteria of the AWGS [3], there were only five in the older group that had reduced grip
strength. The high-salt: low-salt group ratio was 3:2. Furthermore, no decrease in the parameter (SIM)
indicating muscle mass loss could be seen. Therefore, in this study, we can only say that there is a
possibility of sarcopenia risk, and not sarcopenia. Regarding blood tests, the fact that IL-6 and insulin
were significantly higher in the high-salt group may suggest insulin resistance. Insulin resistance
contributes to obesity and aging, and skeletal muscle insulin secretion resistance is involved in the
pathogenesis of sarcopenia. IL-6 has also been shown to induce insulin resistance. Therefore, when
homeostasis model assessment as an index of insulin resistance (HOMA-IR) was calculated [62],
the average value of low salt content and high salt content was 1.18 (0.69):1.49 (0.91), and HOMA-IR
also increased significantly. Increased ALT in blood tests is associated with liver dysfunction. There are
many reports of the risk of sarcopenia due to liver dysfunction, and there are also diagnostic criteria for
liver disease in Japan [4]. Increases in all these parameters are associated with sarcopenia risk [4,12–19].
Next, we investigated the relationship between estimated salt intake, age, and a possibility of
sarcopenia risk. There were 16 parameters with a significant difference. Parameters were stratified into
the younger and older groups, the items that showed a significant difference only in older groups were
the parameters related to fat and the parameters indicating the decrease in muscle strength or physical
function. In addition, only BFP had significantly higher mean values in the high-salt group compared
to the low-salt group in both the younger and older groups. In other words, the results of this study
showed that excessive intake of salt was related with accumulated fat parameters in the bodies in
both younger and older groups. The comparison of muscle strength in this study did not follow
the sarcopenia definition of EWGSOP and AWGS, but HGS/BW, which indicates muscle strength,
and 30CS, which indicates physical function, were significantly decreased in the high-salt group [2–4].
Therefore, in the older group, the high-salt group had fat accumulation and muscle weakness.
The expression of the renalase may also be related to salt intake and sarcopenia [30–32]. However,
we observed no significant relation between blood renalase levels and sarcopenia in this study.
A previous study reported that the daily intake of salt was 18 g, much higher than the 4.0–13.8 g/day
of this experiment [30]. The results of this study did not reveal a link between renalase, salt intake,
and sarcopenia.
Our study compared people with high and low salt intakes and found that those with high-salt
intake had higher fat-related parameters. However, we could not investigate causal relationships and
mechanisms. For example, fat accumulation is associated with a variety of factors. Salt intake can
contribute to fat accumulation. It can also be inferred that fat accumulation changes depend on diet
and physical activity. It cannot be ruled out that factors that have not been measured or considered
may have confounded the observational results in this study. First, a detailed dietary and physical
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Nutrients 2020, 12, 3474
activity survey would be required to elucidate the association with salt intake. A second limitation of
this study is water intake. “Inbody 770”, which measures body composition, is affected by the amount
of water in the body because it uses the bioelectrical impedance analysis method. Therefore, if the
body composition is to be measured strictly, water restriction should be controlled from the previous
day. The third, this study recruited adults over the age of 20, participants ranging from 22 to 81 years
old, with an average age of 56 years. Further recruitment of participants over the age of 60 or 65 should
have been recruited to investigate the association between salt intake and sarcopenia.
Our study does not directly show that excessive salt intake causes sarcopenia, but a diet with
excessive salt is associated with fat accumulation and muscle weakness. Furthermore, aging without
improving diet can lead to the development of sarcopenia. In addition, past papers have reported that
skeletal muscle mass and skeletal muscle strength are maximized in the 20s and 30s [63], and there is a
standard that the target age for sarcopenia is 60 or 65 years [3]. On the other hand, there are reports
targeting people over 40 years old [64]. Controlling the diet from a young age can prevent various
diseases, including the prevention of sarcopenia.
5. Conclusions
This study analyzed the relationship between salt intake and the risk of sarcopenia in the Japanese
population. As a result, the parameters related to obesity were significantly increased and the
parameters related to muscle strength were significantly decreased in the group of high-salt intake
compared with the low-salt intake group. In addition, the results were more significant in the older
group than the younger group. Excessive salt intake may be associated with risk of sarcopenia,
although further analysis is needed.
Author Contributions: Y.Y. and K.K. conceived of and designed the research; Y.Y., K.K., M.M., M.Y., K.A. and T.K.
performed the experiments; Y.Y., K.K., M.M. and M.Y. analyzed the data; Y.Y., K.K., T.S., R.M., M.K., K.Y., S.M. and
K.T. interpreted the results of the experiments; Y.Y. and T.S. prepared the figures; Y.Y. drafted the manuscript; Y.Y.,
K.K., T.S., M.M., M.Y., K.A., T.K., R.M., M.K., K.Y., S.M. and K.T. edited and revised the manuscript. All authors
have read and agreed to the published version of the manuscript.
Funding: This work was supported in part by a Grant-in-Aid for Scientific Research KAKENHI from the Ministry
of Education, Culture, Sports, Science, and Technology, Japan (19H03995,18K17975); Y.Y. and K.K., K.K. and M.M.
were recipients of a Grant-in-Aid for Research Fellowships of Japan Society for the Promotion of Science for Young
Scientists (19J01099, 20J20892).
Acknowledgments: We wish to thank the members of S.M.’s laboratory (University of Tsukuba) for their
technical assistance.
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www.mdpi.com ISBN 978-3-0365-4836-4

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Nutrition and

ISBN 978-3-0365-4835-7 (Hbk)

About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Preface to ”Nutrition and Athletic Performance” . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Jorge Pérez-Gómez, Santos Villafaina, José Carmelo Adsuar, Eugenio Merellano-Navarro

Tomáš Hlinský, Michal Kumstát and Petr Vajda

Néstor Vicente-Salar, Guillermo Santos-Sánchez and Enrique Roche

Monique D. Dudar, Emilie D. Bode, Karly R. Fishkin, Rochelle A. Brown, Madeleine M.

Maija Marttinen, Reeta Ala-Jaakkola, Arja Laitila and Markus J. Lehtinen

Received: 22 January 2020; Accepted: 12 February 2020; Published: 15 February 2020

Abstract: Physical activity, particularly high-intensity eccentric muscle contractions, produces

Keywords: natural polyphenols; curcumin; muscle-damaging exercise; anti-inﬂammatory;

Nutrients 2020, 12, 501; doi:10.3390/nu12020501 www.mdpi.com/journal/nutrients

2. Material and Methods

2.1. Search Strategy

2.2. Selection of Articles: Inclusion and Exclusion Criteria

3.1. Selection of Studies

Figure 1. Selection of Studies.

3.2. Characteristics of the Studies

Elite Athletes 1 Study [31]

3.3. Outcome Measures

A. Summary of Studies Included in This Systematic Review.

200 mg curcumin Evidence of muscle

C. Summary of Studies Included in This Systematic Review (Continued)

4.1. Curcumin Supplementation

4.2. Exercise-Induced Muscle Damage (EIMD)

4.2.1. Eﬀect on Muscle Pain

4.2.2. Eﬀect on Muscle Performance

4.2.3. Eﬀect on Muscle Enzyme Activity

4.3. Eﬀect on Inﬂammatory Markers

4.4. Eﬀect on Oxidative Markers

5. Limitations, Strengths, and Future Lines of Research

Received: 23 February 2020; Accepted: 31 March 2020; Published: 2 April 2020

Keywords: Carbohydrate; high-intensity exercise; fatigue

Nutrients 2020, 12, 982; doi:10.3390/nu12040982 www.mdpi.com/journal/nutrients

2. Materials and Methods

2.2. Study Design

2.3. Preliminary Test

9.14m 10m 3.5m

2.4. Experimental Test

Table 1. Characteristics of the sport nutrition bars for a 70 kg participant.

Description Low-GI High-GI

2.5. Dietary and Physical Activity Monitoring

2.6. Statistical Analysis

3.1. Blinding, Order Eﬀects, and Adverse Events

3.2. Glucose and Insulin Responses

3.3. Serum NEFA and Substrate Oxidation

3.4. Skill Performance and Rating of Perceived Exertion

Condition Low-GI High-GI

5. Conclusions and Practical Application

Received: 6 March 2020; Accepted: 14 April 2020; Published: 17 April 2020

Nutrients 2020, 12, 1121; doi:10.3390/nu12041121 www.mdpi.com/journal/nutrients

2. Materials and Methods

2.1. Study Design

2.2. Study Population

Table 1. Clinical characteristics of male and female subjects.

2.3. Race Course

2.4. Running Speed Data Collection and Standardization

%Running speed = (The subject’s running speed) / ((Average of top 5 ﬁnishers’

)HPDOH )HPDOH

)HPDOH )HPDOH

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