Original Investigation | Nutrition, Obesity, and Exercise

Association of Body Weight With Response to Vitamin D Supplementation

and Metabolism
Deirdre K. Tobias, ScD; Heike Luttmann-Gibson, PhD; Samia Mora, MD, MHS; Jacqueline Danik, MD, DrPH; Vadim Bubes, PhD; Trisha Copeland, MS, RD;
Meryl S. LeBoff, MD; Nancy R. Cook, ScD; I-Min Lee, MD, ScD; Julie E. Buring, ScD; JoAnn E. Manson, MD, DrPH

Abstract Key Points

Question Does body weight modify
IMPORTANCE In the Vitamin D and Omega-3 Trial (VITAL), the effects of randomized vitamin D
metabolism and response to vitamin D
supplementation (cholecalciferol), 2000 IU/d, reduced the risk of several health outcomes among
supplementation?
participants with normal, but not elevated, body weights. It was unclear whether weight had any
association with the outcomes of the supplementation. Findings In this cohort study, a subset
including 16515 participants of the VITAL
OBJECTIVE To investigate whether baseline body mass index (BMI) modifies vitamin D metabolism randomized clinical trial, established and
and response to supplementation. novel vitamin D serum metabolite levels
were on average lower at higher body
DESIGN, SETTING, AND PARTICIPANTS VITAL is a completed randomized, double-blind, placebo- mass index. Supplementation increased
controlled trial for the primary prevention of cancer and cardiovascular disease. In the present cohort vitamin D levels less over 2 years at
study, an analysis was conducted in a subset of VITAL participants who provided a blood sample at higher body mass index.
baseline and a subset with a repeated sample at 2 years’ follow-up. VITAL was conducted from July 1,
Meaning Previous trials observed
2010, to November 10, 2018; data analysis for the present study was conducted from August 1, 2021,
reduced efficacy of vitamin D
to November 9, 2021.
supplementation for outcomes of
cancer, diabetes, and others, in subsets
INTERVENTIONS Treatment outcomes of vitamin D, 2000 IU/d, supplementation vs placebo
of participants with higher body mass
associated with clinical and novel vitamin D–related biomarkers by BMI category adjusted for other
index; the findings of this cohort study
factors associated with vitamin D status.
suggest this may be due to a blunted
metabolism and internal dose at higher
MAIN OUTCOMES AND MEASURES Multivariable-adjusted means (SE) or 95% CIs of vitamin
body weights.
D–related serum biomarkers at baseline and follow-up: total 25-hydroxyvitamin D (25-OHD),
25-OHD3, free vitamin D (FVD), bioavailable vitamin D (BioD), vitamin D–binding protein (VDBP),
albumin, parathyroid hormone (PTH), and calcium, and log-transformed as needed. + Invited Commentary
RESULTS A total of 16 515 participants (mean [SD] age, 67.7 [7.0] years; 8371 women [50.7%]; 12420
+ Supplemental content
Author affiliations and article information are
non-Hispanic White [76.9%]) were analyzed at baseline, including 2742 with a follow-up blood
listed at the end of this article.
sample. Before randomization, serum total 25-OHD levels were incrementally lower at higher BMI
categories (adjusted mean [SE]: underweight, 32.3 [0.7] ng/mL; normal weight, 32.3 [0.1] ng/mL;
overweight, 30.5 [0.1] ng/mL; obesity class I, 29.0 [0.2] ng/mL; and obesity class II, 28.0 [0.2]
ng/mL; P < .001 for linear trend). Similarly, baseline 25-OHD3, FVD, BioD, VDBP, albumin, and calcium
levels were lower with higher BMI, while PTH level was higher (all P < .001 for linear trend).
Compared with placebo, randomization to vitamin D supplementation was associated with an
increase in total 25-OHD, 25-OHD3, FVD, and BioD levels compared with placebo at 2 years’
follow-up, but increases were significantly lower at higher BMI categories (all treatment effect
interactions P < .001). Supplementation did not substantially change VDBP, albumin, PTH, or
calcium levels.

(continued)

Open Access. This is an open access article distributed under the terms of the CC-BY License.

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 JAMA Network Open | Nutrition, Obesity, and Exercise Association of Body Weight With Response to Vitamin D Supplementation and Metabolism

Abstract (continued)

CONCLUSIONS AND RELEVANCE In this randomized cohort study, vitamin D supplementation

increased serum vitamin D–related biomarkers, with a blunted response observed for participants
with overweight or obesity at baseline. These longitudinal findings suggest that BMI may be
associated with modified response to vitamin D supplementation and may in part explain the
observed diminished outcomes of supplementation for various health outcomes among individuals
with higher BMI.

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Introduction
Vitamin D is an essential micronutrient relevant to numerous biological processes. It is produced
endogenously as vitamin D3 (cholecalciferol) or obtained from diet or supplements as either vitamin
D3 or vitamin D2 (ergocalciferol). While the importance of vitamin D adequacy for preventing rickets
and osteomalacia is established, accumulating epidemiologic evidence suggests 25-hydroxyvitamin
D (25-OHD) levels may also be relevant for the incidence and progression of cancer1 and
cardiovascular disease.2 In contrast, meta-analyses of randomized clinical trials of vitamin D
supplementation, including the large-scale Vitamin D and Omega-3 Trial (VITAL), have not reported
benefits on the primary end points of cancer or major cardiovascular disease events.3-5 However,
prespecified secondary analyses in VITAL indicated a statistical interaction by baseline body weight,
whereby randomization to vitamin D supplementation vs placebo was associated with a significant
24% lower cancer incidence,3 42% lower cancer mortality,6 and 22% lower incidence of autoimmune
disease7 among participants with normal body weight (body mass index [BMI] <25.0 [calculated as
weight in kilograms divided by height in meters squared]), but no reductions among those with
overweight or obesity. Moreover, 2 meta-analyses of randomized clinical trials of vitamin D
supplementation and risk of type 2 diabetes similarly indicated a modifying association of BMI.8,9
Excess body weight and obesity are consistently characterized by lower 25-OHD blood levels
and a higher prevalence of vitamin D insufficiency and deficiency in cross-sectional studies.10
Evidence from a bidirectional mendelian randomization study implicated obesity as a driver of low
serum 25-OHD levels, rather than the reverse, although reasons for this observation are largely
unknown.11 Furthermore, vitamin D supplementation has not been shown to modify body weight12
or body composition in VITAL participants,13 indicating weight gain precedes alterations in vitamin D
metabolism rather than vice versa. In VITAL, mean serum total 25-OHD levels with active
supplementation at 1 year follow-up were significantly lower for participants with obesity vs normal
weight (38.6 vs 45.9 ng/mL [to convert to nanomoles per liter, multiply by 2.496]), despite receiving
the same dose.3 Hypotheses for diminished response to vitamin D supplementation in obesity
include that a greater amount of vitamin D, a fat-soluble vitamin, is sequestered in adipose tissue,
leaving lower amounts in circulation.14 Volumetric dilution of vitamin D across a larger adiposity mass
has also been posited.15 Alternatively, obesity may suppress hepatic enzyme 25-hydroxylation of
vitamin D to 25-OHD, thereby reducing bioactivity.16 It remains unknown whether the blunted
response of 25-OHD levels with supplementation at higher body weights also extends to other
biomarkers of vitamin D metabolism. If so, diminished vitamin D bioavailability or activity would be
plausible explanations for the limited effectiveness of vitamin D supplementation to reduce cancer
incidence and mortality, type 2 diabetes,17 and other pertinent outcomes at higher BMIs.
We hypothesized that, in addition to differences in circulating total 25-OHD levels with vitamin
D supplementation, obesity would modify other circulating markers of vitamin D bioavailability and
activity. Of total vitamin D in circulation, 85% to 90% is bound to a vitamin D–binding protein
(VDBP). Bioavailable 25-OHD (BioD) is the remaining 10% to 15% vitamin D fraction in circulation
that is not bound to VDBP; BioD is primarily bound to albumin. Free vitamin D (FVD) levels represent
less than 1% of total vitamin D. Identifying subgroups with suboptimal response to supplementation

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 JAMA Network Open | Nutrition, Obesity, and Exercise Association of Body Weight With Response to Vitamin D Supplementation and Metabolism

underscores the potential role for future precision prevention strategies to tailor dosing and
maximize the effectiveness of vitamin D supplementation for chronic disease prevention. We
investigated this aim in a post hoc analysis of the randomized VITAL, with repeated measures at
baseline and 2 years for total vitamin D, FVD, BioD, VDBP, albumin, parathyroid hormone (PTH),
and calcium.

Methods
Study Population
We performed a post hoc analysis in VITAL, a completed randomized, double-blind, placebo-
controlled 2 × 2 factorial trial of vitamin D3 (cholecalciferol), 2000 IU/d, and marine ω-3 fatty acids, 1
g/d, for the primary prevention of cancer and cardiovascular disease (NCT01169259).3 VITAL was
conducted from July 1, 2010, to November 10, 2018; data analysis for the present study was
conducted from August 1, 2021, to November 9, 2021. Eligible participants were men aged 50 years
or older and women aged 55 years or older who were free of cancer and cardiovascular disease at
baseline enrollment. Details of the trial population, results, and blood collection methods were
previously described in detail.18,19 Briefly, after a 3-month placebo run-in period to demonstrate
adherence, participants were randomized in 5-year age groups, using a computer-generated table of
random numbers, to 1 of the 4 treatment combinations. Investigators and participants were blinded
to active vs placebo study pills. Among the 25 871 individuals randomized, there were 16 515 eligible
participants who contributed baseline blood samples before randomization (October 2010 to March
2014) and, among them, 2742 provided a blood sample at 2 years’ follow-up (Figure 1). These
samples were collected via mail-based kits, in-home visits, or phlebotomy centers and shipped
overnight to the Boston laboratory where they were processed and stored at −170 °C within 36
hours of the sample draw. Height and body weight were self-reported on the baseline questionnaire.
In-person visits were conducted for 1054 Boston-area participants who attended the Brigham and
Women’s Hospital in Boston for detailed phenotyping and biospecimen collections at randomization
and again at 2 years’ follow-up. If participants provided blood samples for both the in-person and
mail-based collections, we analyzed the in-person values. Participants provided written informed
consent at enrollment and the study protocol was approved by the Partners Institutional Review
Board, Boston, Massachusetts. Participants received financial compensation as part of the original
trial. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology
(STROBE) reporting guideline for observational studies.

Figure 1. Flowchart Diagram of Eligible Vitamin D and Omega-3 Trial (VITAL) Participants Included in Analyses

25 871 Randomized VITAL participants

16 908 Baseline blood collection participants

394 Excluded
7 No biomarker data
387 No baseline BMI

16 515 Baseline vitamin D analysis

15 462 Main blood collections
1053 In-person CTSC participants

2742 Year 2 vitamin D analysis

1778 Main blood collections BMI indicates body mass index; CTSC, Clinical and
964 In-person CTSC participants
Translational Science Center.

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Biomarker Assays and Quality Control

Baseline and follow-up samples were shipped and assayed in tandem to avoid systematic error, and
personnel were blinded to collection and randomized treatment status. Total 25-OHD and 25-OHD3
levels were quantified with liquid chromatography tandem mass spectrometry (Quest Diagnostics
Nichols Institute).13 Free vitamin D levels were measured using an enzyme-linked immunoassay and
polyclonal VDBD (Future Diagnostics Solutions BV). We derived BioD, defined as circulating 25-OHD
not bound to VDBP, with the validated equation by Powe et al20 (eMethods in Supplement 1).
Albumin levels (Beckman Coulter Clinical Chemistry AU analyzer; Quest Diagnostics Nichols Institute)
and intact PTH, using a chemiluminescent-based assay (Beckman Coulter), were measured, and
serum calcium levels were quantified via spectroscopy (Quest Diagnostics Nichols Institute).
Coefficients of variation were calculated from blinded duplicate aliquots of 20 participant samples:
total 25-OHD = 4.9%, 25-OHD3 = 4.1%, FVD = 6.8%, VDBP = 15.7%, PTH = 7.6%, and
calcium = 2.0%. VITAL participated in the vitamin D standardization program of the Centers for
Disease Control and Prevention.21

Statistical Analysis
Our analyses excluded participants with missing or extreme baseline BMI (BMI<12.0 or ⱖ60.0).
Participants’ baseline characteristics, demographic characteristics, and health status at trial baseline
were stratified by baseline BMI categories of underweight (<18.5), normal weight (18.5-24.9),
overweight (25.0-29.9), obesity class I (30.0-34.9), and obesity class II (ⱖ35.0). For analyses
including repeated biomarkers at 2 years, we combined the underweight and normal weight
categories due to an insufficient sample size for BMI less than 18.5.
To minimize outliers, we winsorized biomarker values at the bottom and top 0.5%, then
inspected the biomarker distributions for normality. BioD, VDBP, PTH, and calcium were log
transformed for analysis to improve normal distribution. We performed multivariable-adjusted
generalized linear regression models with biomarkers as the continuous dependent variable to
calculate the least square mean (SEM) or geometric mean and 95% CI for log-transformed
biomarkers, stratified by baseline BMI category. We modeled continuous BMI to calculate the P value
linear trends. Multivariable models were adjusted for baseline characteristics of age in years
(continuous), sex, prevalent diabetes, pretrial use of vitamin D supplements, smoking status
(current, past, never; not reported [1.3%]), leisure-time physical activity (above vs below
median = 16.6 metabolic equivalent task hours/week; not reported [<1.0%]), self-reported race and
ethnicity, as required by the funding agency and categorized in the electronic health records (Asian or
Pacific Islander, Black, Native American or Alaska Native, non-Black Hispanic, non-Hispanic White,
other/unknown (option indicated by participant with no further breakdown available); not reported
[2.2%]), US geographic region (West, South, Midwest, Northeast), season at blood draw (winter,
spring, summer, fall), and Brigham and Women’s Hospital Clinical and Translational Science Center
subcohort participation. Categorical covariates were modeled with indicator variables for not
reported covariate data, as the amount of not reported or missing data was small.
We generated waterfall plots to visualize the variability among participants for changes in total
serum 25-OHD concentration at 2 years. Means and SEM (or geometric means and 95% CI for
log-transformed biomarkers) of the continuous biomarkers were estimated from multivariable linear
mixed-effects models with maximum likelihood estimation of the covariance parameters with an
unstructured covariance matrix. The treatment effects per BMI category were calculated as the mean
change difference in biomarkers, plus additional adjustment for the baseline biomarker value in an
analysis of covariance.22,23 We tested for heterogeneity via a 2-way multiplicative interaction term of
randomized treatment assignment × BMI added to the main effects model.
In secondary analyses, we estimated the treatment effect at 2 years according to categories of
central obesity restricted to the subset of participants with in-person technician measurements.
Classification of central obesity was defined based on the World Health Organization waist
circumference (WC) for minimal risk (category I) including women less than 80.0 cm and men less

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than 94.0; moderate risk (category II) including women 80.0 to 87.9 cm and men 94.0 to 101.9 cm;
and high risk (category III) including women, 88.0 cm or more and men, 102.0 cm or more. We also
performed a sensitivity analysis among participants with baseline total 25-OHD levels less than 20.0
ng/mL to estimate whether effect modification by BMI persisted among those with initially
insufficient vitamin D levels. We used SAS, version 9.4 (TS1M5) statistical software (SAS Institute Inc)
to perform the analysis, with an a priori level of significance set as P < .05 with 2-sided
hypothesis tests.

Results
Of the 16 908 VITAL participants in the baseline blood collection, 16 515 had vitamin D–related
biomarkers assayed and were eligible for analysis, with mean (SD) age 67.7 (7.0) years (Figure 1).
There were 238 (1.5%) who self-reported Asian or Pacific Islander race andethnicity, 2445 (15.1%) as
Black, 129 (0.8%) Native American or Alaska Native, 589 (3.6%) non-Black Hispanic, 12420 (76.9%)
non-Hispanic White, and 333 (2.1%) identifying as other or unknown (with no available further
breakdown of category). Baseline characteristics according to BMI category are summarized in
Table 1. A total of 8371 women (50.7%) and 8144 men (49.3%) were included in the analysis.
Participants with overweight (6688 [40.5%]) or obesity (4457 [27.0%]) were, on average, younger
and more likely to self-report Black race and ethnicity, a lower household annual income, and a lower
achieved educational level than participants with normal body weight. Participants who were obese
were less likely to be physically active or report alcohol intake, but smoking status was similar across
BMI categories. We observed similar findings by BMI category for the subset of participants included
in the 2-year change analyses (eTable 1 in Supplement 1).
Mean (SD) serum 25-OHD level before the use of study pills was 30.6 (9.5) ng/mL and most
levels (14 279 [87.5%]) were sufficient at 20.0 ng/mL or greater. Total 25-OHD levels were
significantly lower among participants at higher BMI categories, even with adjustment for other
factors associated with serum vitamin D levels (Table 2); mean (SE) for underweight (32.3 [0.7]
ng/mL), normal weight (32.3 [0.1] ng/mL), overweight (30.5 [0.1] ng/mL), obesity class I (29.0 [0.2]
ng/mL), and obesity class II (28.0 [0.2] ng/mL) (P < .001). Statistically significant inverse trends were
also observed with lower baseline levels of 25-OHD3, FVD, BioD, VDBP, albumin, and calcium at
higher BMI, and higher PTH levels with higher BMI.
Among the 2742 participants with repeated blood collections at year 2, there was a significant
mean (SD) 11.9 (8.6) ng/mL increase in serum 25-OHD level in the group randomized to vitamin D
supplementation, compared with little change in the placebo group (−0.7 [7.9] ng/mL). Waterfall
plots (eFigure in Supplement 1) illustrate the variability of within-person change in total 25-OHD level
according to randomized treatment. The multivariable-adjusted means at baseline and 2 years for all
biomarkers by treatment group and BMI category are given in Figure 2 and Table 3. There were
significant increases in the mean total 25-OHD, 25-OHD3, FVD, and BioD levels at 2 years among
participants randomized to active supplementation, and little or no changes observed for the
placebo group. Furthermore, we observed statistically significant interactions (all interactions
P < .001) by baseline BMI category for these treatment effects vs placebo, whereby the magnitudes
of biomarker increases were lower at higher baseline BMI. For example, for total 25-OHD level, the
mean (SD) increases at 2 years for supplementation vs placebo across BMI strata were 13.5 (0.6)
ng/mL for less than 25.0, 12.7 (0.5) ng/mL for 25.0 to 29.9, 10.5 (0.7) ng/mL for 30.0 to 34.9, and 10.0
(1.0) ng/mL for greater than or equal to 35.0 ng/mL. In contrast, there was no significant difference
with vitamin D supplementation vs placebo in VDBP, albumin, PTH, or calcium levels, and the lack of
association was observed for all BMI categories.
We repeated the above models of treatment effect heterogeneity by baseline WC categories for
the subset of 964 Clinical and Translational Science Center participants with in-person
measurements (eTable 2 in Supplement 1). As with BMI-derived cut points, we observed significant
increases in total 25-OHD, 25-OHD3, FVD, and BioD levels with supplementation vs placebo that

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Table 1. Characteristics of 16 515 VITAL Participants Included in Biomarker Analysis by BMI at Study Baseline

Baseline randomization, No. (%)

Underweight, Normal, Overweight, Obesity I, Obesity II,
Characteristic BMI <18.5 BMI 18.5-24.9 BMI 25.0-29.9 BMI 30.0-34.9 BMI ≥35.0
No. 149 5221 6688 2851 1606
BMI, mean (SD) 17.4 (1.2) 22.8 (1.6) 27.3 (1.4) 32.1 (1.4) 39.8 (4.6)
Age, mean (SD), y 70.6 (7.0) 68.9 (7.1) 67.8 (6.9) 66.7 (6.6) 65.4 (6.3)
Sex
Male 33 (22.1) 2294 (43.9) 3896 (58.3) 1374 (48.2) 547 (34.1)
Female 116 (77.9) 2927 (56.1) 2792 (41.7) 1477 (51.8) 1059 (65.9)
Race and ethnicity
Asian or Pacific Islander 4 (2.8) 141 (2.8) 79 (1.2) 9 (0.3) 5 (0.3)
Black 11 (7.6) 420 (8.2) 892 (13.6) 605 (21.7) 517 (33.0)
Native American or 0 29 (0.6) 47 (0.7) 35 (1.3) 18 (1.1)
Alaska Native
Non-Black Hispanic 3 (2.1) 141 (2.8) 262 (4.0) 115 (4.1) 68 (4.3)
Non-Hispanic White 122 (84.7) 4279 (83.6) 5124 (78.4) 1959 (70.3) 936 (59.7)
Other or unknowna 4 (2.8) 106 (2.1) 135 (2.1) 64 (2.3) 24 (1.5)
Highest educational level
No high school 3 (2.0) 21 (0.4) 57 (0.9) 26 (0.9) 52 (3.2)
High school 10 (6.8) 333 (6.4) 591 (8.8) 351 (12.4) 248 (15.5)
College 57 (38.8) 2001 (38.4) 2734 (40.9) 1327 (46.7) 742 (46.3)
Postcollege 77 (52.4) 2859 (54.8) 3300 (49.4) 1139 (40.1) 560 (35.0)
Exercise, mean (SD), 27.4 (28.9) 28.8 (26.8) 23.8 (24.2) 17.9 (21.8) 12.4 (22.5)
MET-h/wk
Smoking
Never 80 (54.4) 2822 (54.7) 3310 (50.2) 1407 (50.0) 824 (52.2)
Past 47 (32.0) 2016 (39.1) 2928 (44.4) 1254 (44.5) 642 (40.7)
Current 20 (13.6) 324 (6.3) 359 (5.4) 155 (5.5) 113 (7.2)
Alcohol intake
Never 37 (25.2) 1280 (24.9) 1715 (26.0) 1020 (36.4) 769 (48.7)
<1/wk 10 (6.8) 314 (6.1) 451 (6.8) 258 (9.2) 144 (9.1)
1-6/wk 64 (43.5) 1873 (36.4) 2422 (36.7) 950 (33.9) 481 (30.5)
Daily 36 (24.5) 1681 (32.7) 2012 (30.5) 575 (20.5) 185 (11.7)
Diabetes 10 (6.7) 305 (5.8) 774 (11.6) 621 (21.8) 560 (34.9)
Hypertension medication 39 (26.2) 1865 (35.7) 3368 (50.4) 1815 (63.6) 1237 (77.0)
Cholesterol-lowering 29 (19.5) 1521 (29.1) 2799 (41.9) 1278 (44.8) 761 (47.4)
medication
Pretrial supplemental 57 (38.3) 2472 (47.3) 3655 (54.7) 1682 (59.0) 1016 (63.3)
vitamin D
Geographic region
West 43 (28.9) 1065 (20.4) 1174 (17.6) 426 (14.9) 182 (11.3)
South 43 (28.9) 1789 (34.3) 2461 (36.8) 1102 (38.7) 662 (41.2)
Midwest 27 (18.1) 1040 (19.9) 1385 (20.7) 686 (24.1) 438 (27.3)
Northeast 36 (24.2) 1327 (25.4) 1668 (24.9) 637 (22.3) 324 (20.2)
Season at blood draw
Winter 22 (14.8) 928 (17.8) 1296 (19.4) 603 (21.1) 377 (23.5)
Spring 22 (14.8) 861 (16.5) 1127 (16.9) 541 (19.0) 332 (20.7)
Abbreviations: BMI, body mass index (calculated as
Summer 47 (31.5) 1417 (27.1) 1745 (26.1) 717 (25.2) 412 (25.7) weight in kilograms divided by height in meters
Fall 58 (38.9) 2015 (38.6) 2520 (37.7) 990 (34.7) 485 (30.2) squared); CTSC, Clinical and Translational Science
Provided 2-y repeated 26 (17.4) 724 (13.9) 1072 (16.0) 547 (19.2) 373 (23.2) Center; MET, metabolic equivalent of tasks; VITAL,
blood sample Vitamin D and Omega-3 Trial.
CTSC substudy 12 (8.1) 359 (6.9) 447 (6.7) 160 (5.6) 75 (4.7) a
Defined as the option indicated by participant with
participation
no further breakdown available.

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 JAMA Network Open | Nutrition, Obesity, and Exercise Association of Body Weight With Response to Vitamin D Supplementation and Metabolism

were blunted at the higher WC-derived obesity categories. For example, vitamin D supplementation
vs placebo was associated with a mean (SD) 13.1 (0.9) ng/mL greater increase in total 25-OHD level
for participants in the lowest WC category, compared with 12.3 (1.0) ng/mL for the WC central obesity
category II and 11.6 (0.6) ng/mL for the WC central obesity category III (P < .001 for interaction).
Supplementation was not related to changes in VDBP, albumin, PTH, or calcium levels in this subset,
nor did these associations differ by WC category.
In a sensitivity analysis, we evaluated treatment effect heterogeneity by baseline BMI,
restricted to 377 (14.6%) participants with low vitamin D levels at baseline (total 25-OHD <20.0
ng/mL) (eTable 3 in Supplement 1). The overall trends in these findings were like the overall analysis;
however, statistical power for specific between-group comparisons was limited.

Discussion
Our analysis of older US adults from the randomized VITAL presents novel hypothesis-generating
evidence that adiposity blunts the response to vitamin D supplementation. Consistent with prior
cross-sectional evidence, higher overall and central obesity were associated with significantly lower
serum total 25-OHD levels in our large sample size of 16 515 participants at baseline, controlling for
numerous variables used to estimate vitamin D levels. Furthermore, treatment with vitamin D3,
2000 IU/d, supplementation vs placebo significantly increased total 25-OHD, 25-OHD3, FVD, and
BioD levels at 2 years. However, the magnitude of response to supplementation was notably lower
among those with higher BMI, including reduced increases in total 25-OHD levels, as well as novel
markers of circulating vitamin D activity. Thus, increases in vitamin D availability and bioactivity
achieved with supplementation may be modestly or moderately diminished with excess adiposity.
Our innovative longitudinal analyses adjusted for other factors and potential confounders of vitamin
D status change. These trends were also observed among the subgroup of participants with low
vitamin D levels at baseline, suggesting that, even when insufficient for vitamin D, obesity may still
blunt the response to supplementation. There was, however, no associations between supplemental
vitamin D, overall or by BMI, and the vitamin D carrier proteins VDBP or albumin. Parathyroid
hormone and calcium levels were also unaffected by supplementation vs placebo, regardless of BMI
category. Collectively, this evidence warrants further investigation into impaired pharmacokinetics
or efficacy of vitamin D supplementation for populations with higher BMI.

Table 2. Multivariable-Adjusted Mean or Geometric Mean Vitamin D–Related Biomarker Concentrations at Baseline, by BMIa

BMI at baseline randomization, multivariable-adjusted mean (SEM) [N = 16 516]

P value for
Characteristic No. <18.5 18.5-24.9 25.0-29.9 30.0-34.9 ≥35.0 continuous BMI
Total 25-OHD, ng/mL 16 375 32.6 (0.7) 32.4 (0.1) 30.6 (0.1) 29.0 (0.2) 28.0 (0.2) <.001
25-OHD3, ng/mL 16 369 31.8 (0.7) 31.9 (0.1) 30.1 (0.1) 28.5 (0.2) 27.2 (0.2) <.001
Free vitamin D, pg/mL 5175 6.48 (0.27) 6.39 (0.04) 5.93 (0.04) 5.65 (0.06) 5.45 (0.09) <.001
Bioavailable D, geometric mean (95% CI), 5168 2.3 (2.1-2.5) 2.4 (2.3-2.4) 2.2 (2.2-2.2) 2.1 (2.0-2.1) 1.9 (1.9-2.0) <.001
ng/mL
VDBP, mg/mL 5175 467 (442-494) 472 (467-476) 462 (459-466) 452 (446-458) 455 (447-464) <.001
Albumin, g/dL 16 470 4.43 (0.02) 4.37 (0.00) 4.35 (0.00) 4.31 (0.01) 4.22 (0.01) <.001
PTH, geometric mean (95% CI), pg/mL 16 419 33.5 (31.3-35.8) 33.3 (32.9-33.7) 35.7 (35.4-36.1) 37.8 (37.3-38.4) 40.3 (39.4-41.1) <.001
Calcium, geometric mean (95% CI), mg/dL 15 536 9.40 (9.34-9.47) 9.40 (9.39-9.41) 9.38 (9.37-9.39) 9.35 (9.34-9.37) 9.30 (9.28-9.32) <.001

Abbreviations: BMI, body mass index (calculated as weight in kilograms divided by status (never, past, current, not reported), total physical activity (above, below
height in meters squared); 25-OHD, 25-hydroxyvitamin D; PTH, parathyroid hormone; median = 16.6 MET-h/wk), race and ethnicity (Asian or Pacific Islander, Black, Native
VDBP, vitamin D binding protein. American or Alaska Native, non-Black Hispanic, non-Hispanic White, other/unknown,
SI conversion: To convert albumin to grams per liter, multiply by 10; calcium to millimoles not reported), geographic region (West, South, Midwest, Northeast), season of blood
per liter, multiply by 0.25; 25-OHD to nanomoles per liter, multiply by 2.496; PTH to draw (winter, spring, summer, fall), and in-person Clinical and Translational Science
nanograms per liter, multiply by 1. Center subgroup participation.
a
Values were adjusted for baseline factors: age (continuous), sex (male, female),
prevalence of type 2 diabetes, preintervention vitamin D supplement use, smoking

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Figure 2. Multivariable-Adjusted Mean Vitamin D–Related Biomarker Concentrations at Baseline and 2 Years’ Follow-up, by Randomized Treatment Assignment
and Baseline Body Mass Index (BMI)

A Total 25-OHD B 25-OHD3 C Free vitamin D

50 50 12

10
40 40

Free vitamin D, pg/mL

Total 25-OHD, ng/mL

25-OHD3, ng/mL
30 30

20 20
4

10 10
2

0 0 0
Baseline Year 2 Baseline Year 2 Baseline Year 2

D Bioavailable vitamin D E Vitamin D binding protein F Albumin

5 500 5
Vitamin D binding protein, mg/mL

4
Bioavailable vitamin D, ng/mL

450

Albumin, mg/mL
3

400 4

350
1

0 300 3
Baseline Year 2 Baseline Year 2 Baseline Year 2

G PTH H Calcium

50 10
Treatment by
BMI status
40 Placebo
Normal
Overweight
Calcium, mg/dL

Obesity I
PTH, pg/mL

30
Obesity II
9 Vitamin D
20 Normal
Overweight
Obesity I
Obesity II
10

0 8
Baseline Year 2 Baseline Year 2

Concentrations shown for total 25-hydroxyvitamin D (25-OHD) (A), 25-OHD3 (B), free week), race and ethnicity (Asian or Pacific Islander, Black, Native American or Alaska
vitamin D (C), bioavailable vitamin D (D), vitamin D–binding protein (E), albumin (F), Native, non-Black Hispanic, non-Hispanic White, other/unknown, not reported),
parathyroid hormone (PTH) (G), and calcium (H). Circles and bars are least square mean geographic region (West, South, Midwest, Northeast), season of blood draw (winter,
(standard error of the mean) or geometric mean (95% CI) adjusted for baseline factors: spring, summer, fall), and in-person Clinical and Translational Science Center subgroup
age (continuous), sex (male, female), prevalence of type 2 diabetes, preintervention participation. BMI indicates body mass index. To convert albumin to grams per liter,
vitamin D supplement use, smoking status (never, past, current, not reported), total multiply by 10; calcium to millimoles per liter, multiply by 0.25; 25-OHD to nanomoles
physical activity (above, below median = 16.6 metabolic equivalents of task hours per per liter, multiply by 2.496; PTHe to nanograms per liter, multiply by 1.

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Table 3. Multivariable-Adjusted Mean or Geometric Mean Vitamin D–Related Biomarkers at Baseline and 2 Years’ Follow-up, by Randomized Treatment Assignment
and BMIa

Multivariable-adjusted mean (SEM)

Placebo Vitamin D P value for treatment
Treatment effect, effect interaction
b
Biomarker, BMI category Baseline Year 2 Baseline Year 2 mean (SE) by BMI
Total 25-OHD, ng/mL
<25.0 31.7 (0.4) 31.1 (0.5) 31.0 (0.4) 44.0 (0.5) 13.5 (0.6)
25.0-29.9 29.9 (0.4) 28.9 (0.4) 28.7 (0.4) 41.2 (0.4) 12.7 (0.5)
<.001
30.0-34.9 29.0 (0.5) 28.7 (0.6) 28.9 (0.5) 39.4 (0.5) 10.5 (0.7)
≥35.0 28.2 (0.7) 28.3 (0.8) 26.5 (0.6) 37.9 (0.7) 10.0 (1.0)
25-OHD3, ng/mL
<25.0 31.2 (0.4) 30.6 (0.5) 30.8 (0.4) 43.8 (0.5) 13.5 (0.6)
25.0-29.9 29.4 (0.4) 28.1 (0.4) 28.0 (0.4) 40.9 (0.4) 13.4 (0.5)
<.001
30.0-34.9 28.4 (0.5) 27.9 (0.6) 28.1 (0.5) 39.4 (0.6) 11.6 (0.7)
≥35.0 26.4 (0.7) 26.8 (0.8) 25.8 (0.6) 37.6 (0.7) 10.5 (1.0)
Free vitamin D, pg/mL
<25.0 6.13 (0.12) 6.14 (0.16) 6.39 (0.12) 10.20 (0.16) 3.84 (0.19)
25.0-29.9 6.03 (0.10) 6.15 (0.14) 5.77 (0.10) 8.85 (0.14) 2.92 (0.17)
<.001
30.0-34.9 5.75 (0.16) 5.79 (0.20) 5.40 (0.19) 7.79 (0.24) 2.57 (0.32)
≥35.0 5.08 (0.18) 4.96 (0.23) 5.07 (0.17) 7.04 (0.21) 2.22 (0.34)
Bioavailable vitamin D, geometric mean (95% CI), ng/mL
<25.0 2.3 (2.2-2.4) 2.3 (2.2-2.4) 2.4 (2.3-2.5) 3.8 (3.7-4.0) 1.5 (0.1)
25.0-29.9 2.3 (2.2-2.3) 2.3 (2.2-2.3) 2.2 (2.1-2.2) 3.4 (3.2-3.5) 1.2 (0.1)
<.001
30.0-34.9 2.1 (2.0-2.3) 2.1 (2.0-2.3) 2.0 (1.8-2.1) 2.9 (2.7-3.1) 1.0 (0.1)
≥35.0 1.9 (1.7-2.0) 1.9 (1.7-2.0) 1.8 (1.7-2.0) 2.7 (2.5-2.9) 0.9 (0.1)
VDBP, geometric mean (95% CI), mg/mL
<25.0 456 (447-465) 453 (444-461) 451 (443-460) 450 (441-459) 1 (5)
25.0-29.9 449 (441-458) 444 (436-453) 441 (433-449) 441 (433-450) 3 (4)
.56
30.0-34.9 437 (424-449) 429 (415-442) 449 (434-465) 442 (425-460) 0 (10)
≥35.0 432 (414-451) 445 (429-462) 479 (460-498) 482 (466-499) 6 (13)
Albumin, g/dL
<25.0 4.40 (0.01) 4.38 (0.01) 4.39 (0.01) 4.37 (0.01) 0.00 (0.02)
25.0-29.9 4.42 (0.01) 4.38 (0.01) 4.40 (0.01) 4.38 (0.01) 0.02 (0.01)
.39
30.0-34.9 4.39 (0.02) 4.37 (0.02) 4.37 (0.02) 4.34 (0.02) −0.01 (0.02)
≥35.0 4.30 (0.02) 4.26 (0.02) 4.29 (0.02) 4.26 (0.02) 0.00 (0.02)
PTH, geometric mean (95% CI), pg/mL
<25.0 34.2 (32.9-35.6) 34.4 (33.1-35.7) 35.3 (33.9-36.8) 32.7 (31.5-34.0) −2.1 (0.9)
25.0-29.9 36.3 (35.0-37.6) 36.5 (35.2-37.8) 36.3 (35.1-37.6) 33.3 (32.1-34.5) −4.0 (0.8)
.47
30.0-34.9 40.2 (38.3-42.2) 38.0 (36.1-39.9) 36.8 (35.1-38.6) 33.8 (32.2-35.5) −2.0 (1.3)
≥35.0 39.1 (36.4-41.9) 39.1 (36.5-41.8) 41.4 (39.0-44.0) 38.3 (36.1-40.6) −3.6 (1.8)
Calcium, geometric mean (95% CI), mg/dL
<25.0 9.34 (9.30-9.38) 9.29 (9.25-9.33) 9.28 (9.24-9.32) 9.28 (9.24-9.32) 0.01 (0.03)
25.0-29.9 9.34 (9.31-9.38) 9.30 (9.27-9.33) 9.32 (9.28-9.35) 9.33 (9.29-9.36) 0.05 (0.02)
.40
30.0-34.9 9.37 (9.32-9.43) 9.36 (9.30-9.41) 9.42 (9.37-9.48) 9.41 (9.36-9.47) −0.01 (0.04)
≥35.0 9.41 (9.33-9.48) 9.41 (9.34-9.48) 9.38 (9.31-9.45) 9.38 (9.32-9.44) −0.02 (0.05)

Abbreviations: BMI, body mass index (calculated as weight in kilograms divided by American or Alaska Native, non-Black Hispanic, non-Hispanic White, other/unknown,
height in meters squared); 25-OHD, 25-hydroxyvitamin D; PTH, parathyroid hormone; not reported), geographic region (West, South, Midwest, Northeast), season of blood
SEM, standard error of the mean; VDBP, vitamin D binding protein. draw (winter, spring, summer, fall), and in-person Clinical and Translational Science
SI conversion: To convert albumin to grams per liter, multiply by 10; calcium to millimoles Center subgroup participation. Mean treatment effects were additionally adjusted for
per liter, multiply by 0.25; 25-OHD to nanomoles per liter, multiply by 2.496; PTH to baseline biomarker concentrations. Tests for interaction of treatment effect by
nanograms per liter, multiply by 1. baseline BMI category were additionally adjusted for baseline biomarker
a concentrations and baseline biomarker × BMI interaction.
Values were adjusted for baseline factors: age (continuous), sex (male, female),
b
prevalence of type 2 diabetes, preintervention vitamin D supplement use, smoking Sample sizes included for each biomarker: total 25-OHD, n = 2635; 25-OHD3, n = 2632;
status (never, past, current, not reported), total physical activity (above, below free vitamin D, n = 964; bioavailable vitamin D, n = 963; VDBP, n = 964; albumin,
median = 16.6 MET-h/week), race and ethnicity (Asian or Pacific Islander, Black, Native n = 2732; PTH, n = 2730; calcium, n = 1997.

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Several authors of the present study previously explored factors associated with changes in
serum 25-OHD levels from baseline to 1-year follow-up in VITAL and identified minoritized race and
ethnicity, baseline 25-OHD levels, and nonrandomized supplementation as factors related to the
magnitude of change.24 This current examination of an expanded profile of vitamin D metabolites
and biomarkers provides a novel snapshot of vitamin D availability and activity beyond total 25-OHD.
Vitamin D status is typically evaluated with total serum 25-OHD levels, but this composite of bound
and unbound 25-OHD may not accurately reflect the bioactive subfraction (FVD) and may bias
estimates of vitamin D deficiency. Indeed, the main VITAL reported no significant difference in
efficacy of supplementation on cancer incidence by baseline serum 25-OHD levels less than 20.0
ng/mL (hazard ratio [HR], 0.97; 95% CI, 0.68-1.39) vs greater than or equal to 20.0 ng/mL (HR, 0.98;
95% CI, 0.86-1.12). However, there was significant effect modification by baseline BMI for the
association between vitamin D supplementation with cancer incidence. That is, even though most
serum 25-OHD levels were sufficient, vitamin D supplementation was related to a significant
reduction in cancer incidence for BMI less than 25. In contrast, despite having mean lower serum
levels of 25-OHD at baseline, there was no association between vitamin D treatment and cancer
incidence among participants with overweight (HR, 1.04; 95% CI, 0.90-1.21) or obesity (HR, 1.13; 95%
CI, 0.94-1.37).
There are conflicting hypotheses as to why higher BMI might be associated with lower 25-OHD
circulating levels or activity. One theory proposes that, as a fat-soluble vitamin, there is a greater
removal of vitamin D from circulation due to increased storage capacity across higher adiposity
volumes.25,26 Our results are largely consistent with this hypothesis. Evidence from weight loss
interventions also point to vitamin D sequestration as a function of adiposity amount. For example,
the amount of weight loss was associated with greater increases in serum 25-OHD levels among
postmenopausal women during a randomized weight loss intervention, despite no changes in dietary
or supplemental vitamin D intakes.27 Similarly, a cohort of patients undergoing Roux-en-Y gastric
bypass bariatric surgery also had a significant increase in circulating vitamin D levels concurrent with
extreme weight loss, even without receiving vitamin D supplementation.28 Thus, BMI-related
storage capacity and sequestration may contribute to the moderate differences by BMI in circulating
levels of 25-OHD noted with supplementation.
Another hypothesis posits obesity-induced hepatic dysfunction contributes to impaired vitamin
D metabolism. Oral vitamin D enters circulation and is activated enzymatically in the liver to 25-OHD
by cytochrome P450 (CYP) enzymes. Interference in metabolism by obesity and related
cardiometabolic dysfunction would result in a dampened response to vitamin D supplementation on
the amount of circulating 25-OHD and its downstream activity. Animal models demonstrated
downregulation of CYP2R1 in obesity and diabetes.29 It was also shown that surgically induced
weight loss in humans led to increased CYP2R1 activity in adipose tissue.29 Indeed, the effects of
supplementation with vitamin D3, 2000 IU/d, on serum 25-OHD, FVD, and BioD levels were
markedly attenuated across higher BMI categories. Prior evidence for central obesity, reflecting
underlying viscerally accumulated adipose tissue and hepatic fat depots, is mixed. For example, a
cross-sectional analysis in the predominantly White US Framingham Cohort Study observed an
inverse association between serum 25-OHD with standard total computed tomography–derived
quantities of subcutaneous adipose tissue and visceral adipose tissue; however, the investigators did
not report similar inverse trends for 25-OHD with degree of insulin resistance or other obesity-
related cardiometabolic risk factors, independent of adiposity.30 Other studies similarly did not
report associations of total vitamin D status with obesity-related biomarkers independent of the
measures of adiposity.31 In this present analysis, we did not observe further differences in response
by WC. Thus, variability of total serum 25-OHD and 25-OHD3 levels with body weight appears to be
associated with the amount of adiposity per se for some populations, rather than underlying
cardiometabolic health state, although more research is needed in this area.
Parathyroid hormone levels correspond inversely to serum 25-OHD, with insufficient vitamin D
leading to elevated PTH levels and normalization of PTH levels in response to supplementation.32

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The threshold at which vitamin D–induced decreases in PTH levels plateau is hypothesized to reflect
the optimal physiologic concentration of 25-OHD, and this association may differ by obesity status.14
Baseline PTH levels were higher in VITAL participants with higher BMIs; however, changes in PTH
levels with vitamin D supplementation vs placebo did not differ significantly by BMI categories at 2
years’ follow-up. The overall minimal effect on PTH levels is consistent with the study population
being predominantly vitamin D sufficient at baseline. Finally, the vitamin D carrier proteins, VDBP and
albumin, were largely unchanged for all BMI categories, despite increases in total 25-OHD levels with
supplementation.

Limitations
This study has limitations. Our analysis of vitamin D supplementation and biomarkers of availability
and activity of vitamin D was conducted in a large, US-based randomized clinical trial using vitamin
D3, 2000 IU/d, vs placebo, with repeated blood sample collections and long-term follow-up. The
cross-sectional analyses at baseline enrollment were adjusted for potential correlates of BMI and
determinants of vitamin D status, including age, sex, race and ethnicity, season of blood draw,
physical activity, and more. Residual confounding, however, may in part explain the differences by
baseline BMI. The prospective analysis of vitamin D supplementation vs placebo with 2-year changes
in vitamin D–related biomarkers benefits from randomized treatment assignment. Differences in
postrandomization factors related to BMI, such as adherence to study pills or loss to follow-up, may
bias our findings; however, dropout among the subset with repeated blood sample collection was
minimal, and 94% were adherent to taking study pills. It is plausible that factors related to obesity
explain the heterogeneity we observed by BMI category; reassurance is provided by estimates from
multivariable-adjusted models.

Conclusions
In this cohort study providing an explanatory analysis of a large randomized clinical trial, vitamin D3,
2000 IU/d, increased total 25-OHD levels as well as novel markers of vitamin D status, including
25-OHD3, FVD, and BioD, vs placebo at 2 years of intervention. Body mass index status modified the
result of supplementation, with lower response and achieved levels for these biomarkers at higher
BMIs. Vitamin D–binding protein and albumin levels were unchanged with supplementation and
reductions in PTH levels with increased circulating vitamin D levels were consistent across BMI
categories. Sequestering of vitamin D and its metabolites from the circulation into adipose tissue may
contribute to lower serum concentrations and may at least, in part, explain the reduced effectiveness
of supplementation previously reported for cancer end points among VITAL participants with
obesity. Obesity-related pathophysiologic factors, such as impaired vitamin D receptor sensitivity, are
not ruled out and further mechanistic research is warranted. The effective dose to achieve optimal
circulating and bioactive concentrations required for prevention of cancer and diabetes or other
benefits may therefore be higher among patients with excess adiposity, and nutri-kinetics will
elucidate this further.

ARTICLE INFORMATION
Accepted for Publication: October 20, 2022.
Published: January 17, 2023. doi:10.1001/jamanetworkopen.2022.50681
Open Access: This is an open access article distributed under the terms of the CC-BY License. © 2023 Tobias DK
et al. JAMA Network Open.
Corresponding Author: Deirdre K. Tobias, ScD, Brigham and Women’s Hospital, 900 Commonwealth Ave,
Boston, MA 02215 ([email protected]).
Author Affiliations: Division of Preventive Medicine, Department of Medicine, Brigham and Women’s Hospital

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 JAMA Network Open | Nutrition, Obesity, and Exercise Association of Body Weight With Response to Vitamin D Supplementation and Metabolism

and Harvard Medical School, Boston, Massachusetts (Tobias, Luttmann-Gibson, Mora, Bubes, Copeland, Cook,
Lee, Buring, Manson); Department of Nutrition, Harvard T. H. Chan School of Public Health, Boston, Massachusetts
(Tobias); Department of Environmental Health, Harvard T. H. Chan School of Public Health, Boston, Massachusetts
(Luttmann-Gibson); Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s Hospital
and Harvard Medical School, Boston, Massachusetts (Mora); Cardiology Division, Massachusetts General Hospital
and Harvard Medical School, Boston (Danik); Division of Endocrinology, Diabetes and Hypertension, Department
of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts (LeBoff);
Department of Epidemiology, Harvard T. H. Chan School of Public Health, Boston, Massachusetts (Cook, Lee,
Buring, Manson).
Author Contributions: Drs Tobias and Manson had full access to all of the data in the study and take responsibility
for the integrity of the data and the accuracy of the data analysis.
Concept and design: Tobias, Danik, Manson.
Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: Tobias.
Critical revision of the manuscript for important intellectual content: All authors.
Statistical analysis: Tobias, Luttmann-Gibson, Cook.
Obtained funding: Mora, Danik, Manson.
Administrative, technical, or material support: Mora, Bubes, Lee, Manson.
Supervision: Danik, Copeland, Manson.
Conflict of Interest Disclosures: Dr Mora reported receiving grant R01HL134811 from the National Institutes of
Health (NIH) National Heart, Blood, and Lung Institute and nonfinancial support in the form of laboratory
measurements from Quest Diagnostics Study during the conduct of the study; personal fees from Pfizer outside
the submitted work. Dr Danik reported receiving grants from the American Heart Association during the conduct
of the study. Dr Cook reported receiving grants from the NIH to the institution during the conduct of the study. Dr
Lee reported receiving grants the NIH during the conduct of the study. Dr Buring reported receiving grants from
the NIH during the conduct of the study, and her spouse was on the scientific advisory board of Pharmavite, which
provided vitamin D and placebo. Dr Manson reported receiving grants from the NIH during the conduct of the
study and grants from the NIH and Mars Edge outside the submitted work. No other disclosures were reported.
Funding/Support: The Vitamin D and Omega-3 Trial was supported by grant R01AT011729 from the National
Center for Complementary and Integrative Health and, during the intervention phase, was supported by grants
U01 CA138962 and R01 CA138962 from the National Cancer Institute; National Heart, Lung, and Blood Institute;
Office of Dietary Supplements; National Institute of Neurological Disorders and Stroke; and the National Center for
Complementary and Integrative Health. The ancillary studies are supported by grants from multiple institutes,
including the National Heart, Lung, and Blood Institute; the National Institute of Diabetes and Digestive and Kidney
Diseases; the National Institute on Aging; the National Institute of Arthritis and Musculoskeletal and Skin Diseases;
the National Institute of Mental Health; and others. Pharmavite LLC of Northridge, California (vitamin D) and
Pronova BioPharma of Norway and BASF (Omacor fish oil) donated the study agents, matching placebos, and
packaging in the form of calendar packs. Quest Diagnostics measured serum 25-hydroxyvitamin D, parathyroid
hormone, and other biomarkers at no cost to the study. Dr LeBoff reported grants from National Institute of
Arthritis and Musculoskeletal and Skin Diseases RO1 AR070854 and grants from National Institute of Arthritis and
Musculoskeletal and Skin Diseases R01 AR059775.
Role of the Funder/Sponsor: The funding institutions had no role in the design and conduct of the study;
collection, management, analysis, and interpretation of the data; preparation, review, or approval of the
manuscript; and decision to submit the manuscript for publication.
Data Sharing Statement: See Supplement 2.
Additional Contributions: We thank the Centers for Disease Control and Prevention (Drs Hubert Vesper and
Julianne Cook Botelho) for their collaboration on the standardization and calibration of the total 25(OH)D
measurements.

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SUPPLEMENT 1.
eMethods. Calculation of Bioactive Vitamin D (BioD)
eTable 1. Characteristics of VITAL Participants Included in Analyses of Treatment Effect at 2 Years
eTable 2. Multivariable-Adjusted Mean (Standard Error) or Geometric Mean (95% CI) Vitamin D–Related
Biomarkers at Baseline and 2 Years Follow-up, by Randomized Treatment Assignment and Baseline WC (cm)
eTable 3. Multivariable-Adjusted Mean (Standard Error) or Geometric Mean (95% CI) Vitamin D–Related
Biomarkers at Baseline and 2 Years Follow-up, by Randomized Treatment Assignment and Baseline Body Mass
Index Restricted to Low Serum Vitamin D (<20.0 ng/mL) at Baseline Randomization
eFigure. Waterfall Plots of Individual Participant Response, as Change in Serum Total 25-OHD Concentrations
(ng/mL), for (A) Placebo and (B) Vitamin D Supplementation at 2 Years Follow-up

SUPPLEMENT 2.
Data Sharing Statement

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