C LIN I CA L R ES E AR CH A RT IC LE

Vitamin D and Testosterone in Healthy Men:

A Randomized Controlled Trial

Elisabeth Lerchbaum,1 Stefan Pilz,1 Christian Trummer,1 Verena Schwetz,1

Oliver Pachernegg,2 Annemieke C. Heijboer,3,4 and Barbara Obermayer-Pietsch1
1
Department of Internal Medicine, Division of Endocrinology and Diabetology, Medical University of Graz,

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8036 Graz, Austria; 2Department of Urology, Medical University of Graz, 8036 Graz, Austria; 3Department
of Clinical Chemistry, Endocrine Laboratory, VU University Medical Center, 1081 HV Amsterdam,
The Netherlands; and 4Laboratory of Endocrinology, Academic Medical Center, 1105 AZ Amsterdam,
The Netherlands

Context: Available evidence shows an association of vitamin D with androgen levels in men.
However, results from preliminary randomized controlled trials (RCTs) are conflicting.

Objective: To evaluate whether vitamin D supplementation increases total testosterone (TT) levels in
healthy men.

Design: The Graz Vitamin D&TT-RCT is a single-center, double-blind, randomized, placebo-

controlled trial conducted between December 2012 and January 2017.

Setting: Endocrine outpatient clinic at the Medical University of Graz, Austria.

Participants: Ninety-eight healthy men with TT levels $10.4 nmol/L and 25-hydroxyvitamin D
[25(OH)D] levels ,75 nmol/L completed the study.

Intervention: Subjects were randomly assigned to receive 20,000 IU/wk of vitamin D3 (n = 50) or
placebo (n = 50) for 12 weeks.

Main Outcome Measures: Primary outcome was TT measured using mass spectrometry. Secondary
outcomes were free testosterone, sex hormone-binding globulin, estradiol, follicle-stimulating
hormone, and luteinizing hormone levels; free androgen index; metabolic characteristics; and
body composition.

Results: In healthy men [mean values 6 standard deviation: age, 39 years (613 years); 25(OH)D level,
53.3 nmol/L (618.3 nmol/L); TT, 19.1 nmol/L (65.6 nmol/l)], no significant treatment effect on TT was
found; however, there were significant effects on quantitative insulin sensitivity check index
(QUICKI) and a trend toward decreased Matsuda index. In the treatment group, median
(interquartile range) changes for TT, QUICKI, and Matsuda index were 0.5 nmol/L (20.63 to
0.63 nmol/L; P = 0.497), 20.02 (20.04 to 0.01; P = 0.034), and 20.9 (23.2 to 0.8; P = 0.051), respectively.

Conclusion: Vitamin D treatment had no effect on TT levels in middle-aged healthy men with normal
baseline TT, but it significantly decreased QUICKI. Additional studies investigating vitamin D effects on TT
and insulin sensitivity in healthy men are required. (J Clin Endocrinol Metab 102: 4292–4302,
2017)

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: 25(OH)D, 25-hydroxyvitamin D; AUCglucose, area under the curve
Printed in USA for glucose; AUCinsulin, area under the curve for insulin; FAI, free androgen index; FSH,
Copyright © 2017 Endocrine Society follicle-stimulating hormone; FT, free testosterone; Graz Vitamin D&TT-RCT, Graz Vitamin
Received 23 June 2017. Accepted 23 August 2017. D and Total Testosterone Randomized Clinical Trial; ID-LC-MS/MS, isotope-dilution liquid
First Published Online 29 August 2017 chromatography tandem mass spectrometry; LH, luteinizing hormone; OGTT, oral glucose
tolerance test; PTH, parathyroid hormone; QUICKI, quantitative insulin sensitivity
check index; RCT, randomized controlled trial; SD, standard deviation; SHBG, sex hormone-
binding globulin; TT, total testosterone; VDR, vitamin D receptor.

4292 https://academic.oup.com/jcem J Clin Endocrinol Metab, November 2017, 102(11):4292–4302 doi: 10.1210/jc.2017-01428
doi: 10.1210/jc.2017-01428
itamin D deficiency is considered an important
V public health problem (1). Beyond the well-known
relationship between vitamin D deficiency and muscu-
loskeletal diseases, evidence is accumulating that vitamin
D deficiency is also a risk marker for insulin resistance (2),
cardiovascular disease (3), infectious and autoimmune
diseases (4), cancer (5), and increased mortality risk (1).
Similarly, several lines of evidence suggest that low tes-
tosterone levels are associated with adverse events in-
cluding higher cardiovascular and all-cause mortality
(6–9). Interestingly, men with combined vitamin D and
androgen deficiencies are at high risk for all-cause and
cardiovascular mortality, suggesting that a parallel de-
ficiency of both hormones is a powerful marker of poor
health (10).
The vitamin D receptor (VDR) regulates about 3% of
the human genome and is almost ubiquitously expressed
in human cells, which underlines the clinical significance
of the vitamin D endocrine system (1, 4, 11). VDR and
vitamin D metabolizing enzymes are concomitantly
expressed in the reproductive male tract, including Leydig
cells (12), suggesting a potential role of vitamin D in the
production of male reproductive hormones. This notion is
supported by an association of vitamin D and androgen
levels in men suggested by previous studies (13–15).
However, several recent studies among young and healthy
men failed to find an association of vitamin D and an-
drogen levels (16, 17). Furthermore, evidence from pre-
liminary randomized controlled trials (RCTs) revealed
conflicting results. We previously found a significant in-
crease of androgen levels after vitamin D supplementation
in obese men undergoing weight reduction (18), whereas
other studies did not find a significant vitamin D effect on
total testosterone (TT) (19, 20).
Therefore, we designed the Graz Vitamin D and Total
Testosterone Randomized Clinical Trial (Graz Vitamin
D&TT-RCT) to investigate the effects of vitamin D
supplementation on TT concentrations in men.
Methods
Study design
The Graz Vitamin D&TT-RCT is a single-center, random-
ized, double-blind, placebo-controlled trial conducted at the
Medical University of Graz, Graz, Austria. The trial was
designed to investigate the effect of vitamin D supplementation
(12 weeks) on TT levels in men. To further investigate a possible
short-term effect of vitamin D treatment, we performed an
additional study visit after 4 weeks of vitamin D supplemen-
tation. The Graz Vitamin D&TT-RCT examines vitamin D
effects in 100 men with normal TT levels as well in 100 men with
low TT levels (recruitment is ongoing; study end expected later
in 2017).
The design, conduction, and publication of this study adhere
to the recommendations of the Consolidated Standards of
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Reporting Trials Statement. The study was performed in ac-
cordance with the Good Laboratory Practice, Good Clinical
Practice guidelines, and the Declaration of Helsinki. The trial
was registered at http://www.clinicaltrialsregister.eu (EudraCT
no. 2011-003575-11) and at clinicaltrials.gov (ClinicalTrials.
gov no. NCT01748370). The study protocol was approved
by the ethics committee of the Medical University of Graz (EK
23-513 ex 10/11) and written informed consent was obtained
from each participant before entering the study.
Subjects
Eligible study participants were men $18 and ,70 years old
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with 25-hydroxyvitamin D [25(OH)D] levels ,75 nmol/L and
TT levels $10.4 nmol/L. To obtain a relatively homoge-
nous group of men with TT levels in the normal range, we
used $10.4 nmol/L as cutoff value, which is the cutoff used
in the Boston Area Community Health Survey (21) to define
hypogonadism but is slightly higher than that used by the local
university laboratory. Exclusion criteria were hypercalce-
mia (serum calcium level .2.65 mmol/L); oral or transdermal
testosterone supplementation in the last 2 months before en-
tering the study; intramuscular testosterone supplementation
6 months before entering the study; regular intake of vitamin D
supplements before study entry; chronic diseases (such as di-
abetes mellitus), thyroid disease, endocrine disturbances in need
of treatment, or diseases known to interfere with vitamin D
intake or sensitive to vitamin D intake (including inflamma-
tory diseases with granuloma, such as sarcoidosis, tuberculosis,
Wegener granulomatosis); other forms of vasculitis and in-
flammatory bowel diseases; intake of medication influencing
metabolic or endocrine parameters (e.g., insulin sensitizers,
insulin, or glucocorticoids) in the last 3 months before study
entry; prostate-specific antigen level .4 ng/mL (or .3 ng/mL
in men at high risk for prostate cancer); palpable prostate nodule
or induration; hematocrit .50%; untreated severe obstructive
sleep apnea; severe lower urinary tract symptoms; uncontrolled
or poorly controlled heart failure; or a history of prostate cancer,
breast cancer, orchidectomy, and chromosomal disorders (e.g.,
Klinefelter syndrome).
Men were recruited from the outpatient clinic of the De-
partment of Internal Medicine, Division of Endocrinology and
Diabetology, and the outpatient clinic of the Department of
Urology, Medical University of Graz, Graz, Austria, as well as
from male hospital staff and male family members of hospital
staff. Men were informed about the trial either by a conver-
sation in the outpatient clinic, by a telephone call, or by written
information posted in the respective outpatient clinics. All
patients were informed that the participation in the study is
voluntary and that refusal to participate as well as stopping at
any time without giving reasons, without any consequences, is
possible. Written informed consent was obtained from all
subjects before carrying out any study-related procedures.
Intervention
Subjects were allocated to either the vitamin D or placebo
group according to a computer-generated randomization list
using a ratio of 1:1. The study medication was placed in
numbered bottles according to the computer-generated ran-
domization list. Randomization procedures were conducted
using web-based software (http://www.randomizer.at/) with
Good Clinical Practice compliance as confirmed by the Austrian
Agency for Health and Food Safety.
4294 Lerchbaum et al Vitamin D and Testosterone in Men
The treatment group received an oral dose of 20,000 IU
vitamin D (equivalent to 2,857 IU/d) as 50 oily drops weekly
(Oleovit D3 drops; Fresenius Kabi Austria, Linz, Austria) for
12 weeks and the placebo group received 50 oily drops without
vitamin D weekly (Fresenius Kabi Austria) for 12 weeks. Pla-
cebo oil contained the same oil as Oleovit D3 drops (without
vitamin D content). All investigators who enrolled participants,
collected data, and assigned intervention were masked to
participant allocation. To improve and verify compliance, pa-
tients were asked to return the study medication bottles (full as
well as empty bottles) at study end (visit 3).
Primary outcome
The primary outcome was the change in TT levels after
12 weeks of vitamin D supplementation compared with placebo.
TT concentrations were assessed at baseline, after 4 weeks, and
after 12 weeks at the end of the study.
Secondary outcomes
The secondary outcomes were the changes in endocrine
parameters, including levels of free testosterone (FT), free an-
drogen index (FAI), sex hormone-binding globulin (SHBG),
follicle-stimulating hormone (FSH), luteinizing hormone (LH),
and estradiol after vitamin D supplementation compared with
those in the placebo group. SHBG concentrations were assessed
at baseline, after 4 weeks, and after 12 weeks (study end),
whereas FSH, LH, and estradiol levels were assessed at baseline
and after 12 weeks.
Further prespecified secondary outcomes include changes in
metabolic parameters [i.e., insulin resistance, insulin sensitivity,
serum lipid levels, area under the curve (AUC) for glucose
(AUCglucose) and for insulin (AUCinsulin), and body compo-
sition (fat mass and lean mass)], as well as sexual, psychological,
and physical symptoms after vitamin D supplementation
(assessed at baseline and after 12 weeks).
There was no change in study outcomes after the trial
commenced. Although prespecified as a secondary outcome,
sexual, psychological, and physical symptoms were assessed
only at baseline, because of an organizational mistake.
Procedures
Basal blood samples for 25(OH)D, parathyroid hormone
(PTH), TT, SHBG, LH, FSH, estradiol, glucose, insulin, lipids,
and calcium were collected between 8:00 AM and 9:00 AM after
an overnight fast. Levels of 25(OH)D and TT measured by
immunoassays were used for evaluation of inclusion criteria.
Biobanking of remaining blood samples was performed by
freezing and storing the samples at 280°C until analysis. Serum
levels of 25(OH)D and TT were additionally measured by
isotope-dilution liquid chromatography tandem mass spec-
trometry (ID-LC-MS/MS) in 2017. 25(OH)D and TT levels
measured by ID-LC-MS/MS were used for statistical ana-
lyses. FT values were calculated from TT (measured by ID-LC-
MS/MS), SHBG, and albumin, according to Vermeulen et al.
(22). The FAI was calculated as TT (nmol/L; measured by
ID-LC-MS/MS) divided by SHBG (nmol/L) 3 100.
All participants underwent a fasting 75-g oral glucose tol-
erance test (OGTT). Blood samples were drawn after 30, 60,
and 120 minutes for determination of glucose and insulin
concentrations. AUCglucose and AUCinsulin were calculated
according to the trapezoidal method. Insulin resistance was
J Clin Endocrinol Metab, November 2017, 102(11):4292–4302
estimated using homeostatic model assessment-insulin re-
sistance and calculated as fasting plasma insulin (mU/mL) 3
fasting plasma glucose (mg/dL) divided by 405. The quanti-
tative insulin sensitivity check index (QUICKI) was used to
estimate insulin sensitivity and calculated as 1/log fasting in-
sulin (mU/mL) plus log fasting glucose (mg/dL) (23). To assess
b-cell function, HOMA-b was calculated as [20 3 fasting
insulin (mU/mL)] divided by [fasting glucose (mmol/L) 2 3.5].
The Matsuda index was calculated as follows: 10,000 divided
by the square root of [(fasting glucose 3 fasting insulin) 3
(mean glucoseOGTT 3 mean insulinOGTT)] (24). Additional
methods are described in the Supplemental Methods.
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Statistical analyses
Sample size calculation was based on the results of our pilot
study (18) showing TT levels [6 standard deviation (SD)] of
10.7 nmol/L (63.9 nmol/L) vs 13.4 nmol/L (64.7 nmol/L) at
baseline and after 1 year of vitamin D treatment, respectively.
We calculated a sample size of 92 patients. The probability is
90% that the study will detect a treatment difference at a two-
sided 0.05 significance level, if the true difference between
treatments is 0.78 nmol/L. This is based on the assumption that
the SD of the response variable is 1.13. To compensate for
subjects leaving during the study, we enrolled 50 patients
per group.
Data are presented as median with interquartile range.
Categorical data are presented as percentages. The distribution
of data was analyzed by descriptive statistics and Kolmogorov-
Smirnov test. Skewed variables were log transformed and
rechecked for normal distribution. Because not all parameters
had a normal distribution even after log transformation, we
used parametric and nonparametric tests for group compari-
sons. Student t, Mann-Whitney U, and x2 tests were used for
comparisons of baseline characteristics between the vitamin D
and placebo groups.
To study seasonal variation, we subdivided the year into
3-month measurement periods—January through March
(season 1), April through June (season 2), July through Sep-
tember (season 3), and October through December (season 4)—
to address the seasonal changes in availability of sunlight.
Seasonal variation (stratified by month as well as by season) of
25(OH)D, PTH, TT, and FT levels, and of FAI was analyzed by
analysis of variance.
Analyses of primary and secondary outcome variables were
performed according to the intention-to-treat principle and
inclusion of all participants with baseline and follow-up values
of the respective outcome variable. We did not perform data
imputation for missing values. A paired Student t test and
Wilcoxon test were used for comparisons of outcome variables
at baseline and follow-up. Changes between baseline and study
end were calculated as the difference between the study end and
baseline values of outcome variables. Student t test and the
Mann-Whitney U test were used for comparisons of the change
in values between the vitamin D and placebo groups. All sta-
tistical procedures were performed with SPSS version 23 (IBM,
Armonk, NY). P , 0.05 was considered statistically significant.
Results
Blood samples were taken from approximately 500 men
and evaluated for TT levels and 25(OH)D levels (Fig. 1).
doi: 10.1210/jc.2017-01428
Figure 1. Study flowchart showing recruitment, dropout, and follow-up of study participants.
Men with 25(OH)D levels ,75 nmol/L, TT levels $10.4
nmol/L, and a medical history without any exclusion
criteria were informed about the study, its purpose,
potential benefits, and possible risks, and were invited to
participate in the trial. Main reasons for exclusion were
25(OH)D levels .30 ng/mL, TT level ,10.4 nmol/L, as
well as refusal to participate. A total of 100 men who
met all inclusion and exclusion criteria and who gave
their written informed consent were enrolled in the study
and randomly assigned to the treatment or placebo
group. The first subject was randomly assigned in De-
cember 2012 and the last follow-up was performed in
January 2017. Baseline characteristics of all study par-
ticipants are shown in Table 1. We found no significant
difference in baseline characteristics between the vitamin
D and placebo groups. Most subjects were recruited in
seasons 1 and 2. No subject reported the intake of calcium
supplements. The mean (6SD) overall treatment period
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was 89 6 8 days in the vitamin D group and 87 6 7 days
in the placebo group. A total of 98 men completed the
study (Fig. 1) and were analyzed for primary and sec-
ondary outcomes.
Seasonal variation
When stratified by month, we found a significant
difference in 25(OH)D (P , 0.001) and PTH (P = 0.035)
levels (data not shown). Furthermore, when stratified by
season, we observed a significant difference in 25(OH)D
levels and a trend toward different PTH levels (Supple-
mental Table 1). No significant seasonal variation
(stratified by month and season) was seen for TT or FT
levels, or FAI.
Endocrine characteristics
In the vitamin D group, we found a significant increase
in estradiol levels, a significant decrease in SHBG levels,
4296 Lerchbaum et al Vitamin D and Testosterone in Men J Clin Endocrinol Metab, November 2017, 102(11):4292–4302

Table 1. Baseline Characteristics of Study Participants

All Study Participants (N = 100) Vitamin D Group (n = 50) Placebo Group (n = 50)

Median IQR Median IQR Median IQR P Value

Age, y 37 27–50 34 26–48 38 28–52 0.256
BMI, kg/m2 25.1 22.8–26.8 25.0 22.2–26.5 25.2 23.1–27.8 0.791
25(OH)D, nmol/L 52 42–66 52 42–65 51 43–68 0.596
PTH, pg/mL 46.2 36.0–54.6 49.5 37.0–55.6 44.5 35.8–50.2 0.504
Serum calcium, mmol/L 2.40 2.35–2.42 2.38 2.33–2.42 2.40 2.36–2.43 0.356
Urine calcium, mmol/L 2.61 1.56–3.88 2.65 1.66–3.88 2.49 1.33–4.00 0.521
TT, nmol/L 18.0 15.8–21.5 18.7 15.8–22 17.7 15.6–21.1 0.333

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FT, ng/mL 0.096 0.079–0.113 0.098 0.081–0.118 0.096 0.077–0.111 0.450
SHBG, nmol/L 40.2 31.1–53.8 40.6 30.1–49.4 39.8 31.3–55.7 0.934
FAI 7.4 5.0–11.6 7.6 5.1–12.3 7.0 4.7–10.6 0.560
Estradiol, ng/mL 32.3 25.1–42.4 32.3 24.2–39.1 32.6 26.4–48.6 0.475
FSH, mU/mL 4.5 3.0–6.2 4.8 3.2–6.3 4.4 2.9–5.1 0.491
LH, mU/mL 3.7 2.8–5.1 3.7 2.6–5.1 3.5 2.9–5.1 0.705
HOMA-IR 1.7 1.02.5 1.5 1.0–2.4 2.0 1.1–2.5 0.291
HOMA-b 118.9 82.4–172.1 116.5 74.3–165.4 126.4 86.5–182.4 0.509
Matsuda index 6.7 4.5–9.8 7.1 4.6–10.5 6.2 4.3–9.3 0.358
QUICKI 0.35 0.33–0.38 0.36 0.33–0.39 0.34 0.33–0.36 0.291
AUCglucose 205.8 176.3–232.5 204.5 171.3–231.3 205.8 176.5–234.3 0.828
AUCinsulin 79.0 54.3–118.3 76.4 54.3–112.9 79.3 54.8–123.9 0.704
Total cholesterol, mg/dL 189 164–216 187 163–209 190 171–218 0.481
HDL-C, mg/dL 60 51–69 61 51–72 57 51–68 0.238
LDL-C, mg/dL 107 86–127 101 85–127 113 93–130 0.388
Triglycerides, mg/dL 92 62–133 83 60–123 99 65–146 0.209
Fat mass, kg 19.8 14.5–24.9 18.7 14.2–23.1 21.7 13.9–23.0 0.700
Lean mass, kg 58.6 54.9–62.2 58.6 55.7–61.9 58.6 54.4–62.4 0.465
AMS score 22 19–26 23 19–26 22 19–25 0.673
IIEF-EF score 30 28–30 30 27–30 30 28–30 0.999
Season, %a
1 39 40 38 0.541
2 34 30 38
3 10 14 6
4 17 16 18

Comparisons of baseline characteristics between men in vitamin D and placebo group were performed using Student t, Mann-Whitney U, and x2 tests.
Abbreviations: AMS, Aging Male’s Symptoms questionnaire; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; IIEF-EF,
International Index of Erectile Function-Erectile Function questionnaire.
a
Season 1: January through March; season 2: April through June; season 3: July through September; season 4: October through December.

and no significant change in the remaining endocrine Mineral metabolism

parameters (Tables 2 and 3). In the placebo group, we
found no significant change in TT, FT, SHBG, estradiol,
FSH, or LH levels, or FAI. We observed no significant
treatment effects on endocrine parameters.
Metabolic characteristics
In the vitamin D group, we found a significant
increase of homeostatic model assessment-insulin re-
sistance levels as well as a significant decrease in the
Matsuda index and QUICKI (Table 2). In the placebo
group, we found no significant change in metabolic
parameters, or fat or lean mass. The QUICKI decreased
significantly [P = 0.034; Fig. 2(a)], and there was a trend
toward decreased Matsuda index in the vitamin D group
[P = 0.051; Fig. 2(b)]. We observed no significant
treatment effect on the remaining metabolic parameters,
or fat and lean mass.
Parameters of mineral metabolism are shown in
Table 4. We found a significant difference in the change in
25(OH)D and PTH values between treatment groups,
whereas no significant difference was observed in the
change in serum calcium or urine calcium levels.
No important harms or unintended treatment effects
were observed during the study. Furthermore, no study
participant treated with vitamin D had developed hy-
percalcemia at the final study visit.
Despite careful verification of inclusion and exclusion
criteria, one study participant with a TT concentra-
tion ,10.4 nmol/L was included by mistake, and ran-
domly assigned to the vitamin D group and treated. By
adhering to the intention-to-treat principle, we included
this study participant in our analyses. When performing a
sensitivity analysis excluding this patient, our results
remained materially unchanged.
doi: 10.1210/jc.2017-01428 https://academic.oup.com/jcem 4297

Table 2. Outcome Variables at Baseline and Study End, and Changes From Baseline
Baseline Visit Study End Change From Baseline to Study Enda Within-Group
Differences
b
Median IQR Median IQR Median IQR P Value P Valuec
Endocrine characteristics
TT, nmol/L
Vitamin D (n = 49) 18.7 15.9–21.9 19.4 17.2–21.2 0.5 22.2 to 2.2 0.497 0.922
Placebo (n = 49) 17.6 15.7–20.5 18.3 15–21.4 0.5 21.9 to 2.6 0.424
FT, ng/mL
Vitamin D (n = 48) 0.100 0.085–0.108 0.102 0.009–0.131 0 20.02 to 0.01 0.690 0.112
Placebo (n = 49) 0.096 0.079–0.110 0.095 0.075–0119 0 20.01 to 0.01 0.186
SHBG, nmol/L

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Vitamin D (n = 48) 39.9 30.1–48.4 37.9 31.1–47.1 22.5 23.05 to 5.6 0.657 0.028
Placebo (n = 49) 39.7 31.2–54.1 39.5 30.1–48.6 21.2 24.4 to 7.8 0.257
FAI
Vitamin D (n = 48) 7.6 5.1–11.8 7.4 5.5–9.6 20.39 21.67 to 1.08 0.655 0.242
Placebo (n = 49) 6.9 4.9–10.0 6.8 5.0–9.6 0.09 21.95 to 1.22 0.561
Estradiol, ng/mL
Vitamin D (n = 48) 32.3 23.8–38.8 35.3 27.2–43.4 3.2 25.2 to 12.4 0.448 0.035
Placebo (n = 49) 30.7 25.3–44.0 37.3 30.1–48.6 5.6 28.8 to 11.3 0.269
FSH, mU/mL
Vitamin D (n = 48) 4.8 3.0–6.3 4.1 3–5.8 20.04 20.91 to 0.8 0.827 0.630
Placebo (n = 49) 3.8 2.7–5.1 4.3 2.9–6.4 0.43 20.68 to 1.07 0.244
LH, mU/mL
Vitamin D (n = 48) 3.7 2.4–4.9 3.3 2.8–4.5 20.19 20.91 to 0.8 0.722 0.886
Placebo (n = 49) 3.3 2.8–4.4 3.3 2.5–4.4 20.16 21.23 to 1.04 0.516
Metabolic characteristics
HOMA-IR
Vitamin D (n = 47) 1.4 1.0–2.4 2.0 1.4–2.8 0.42 20.26 to 1.01 0.084 0.020
Placebo (n = 48) 2.0 1.2–2.5 1.8 1.3–2.5 0.07 20.65 to 0.56 0.999
HOMA-b
Vitamin D (n = 47) 105.7 69.3–155.5 125.9 91.2–183.8 14.6 226.3 to 56.3 0.517 0.141
Placebo (n = 48) 126.4 88.8–172.1 124.9 94.5–173.6 15.2 233.9 to 46.9 0.460
Matsuda index
Vitamin D (n = 45) 7.6 4.8–10.5 6.6 4.2–8.8 20.9 23.2 to 0.8 0.051 0.037
Placebo (n = 48) 6.1 4.3–8.5 6.2 4.6–9.7 0.2 21.4 to 2.3 0.418
QUICKI
Vitamin D (n = 47) 0.37 0.34–0.39 0.34 0.33–0.36 20.02 20.04 to 0.01 0.034 0.012
Placebo (n = 48) 0.34 0.33–0.36 0.35 0.33–0.37 0.00 20.02 to 0.02 0.975
AUCglucose
Vitamin D (n = 48) 204.6 177.3–231.2 214.9 192.9–238.0 8.0 19.5 to 27.8 0.237 0.127
Placebo (n = 48) 205.8 176.4–234.3 205.8 186.8–230.0 1.0 236.6 to 32.1 0.560
AUCinsulin
Vitamin D (n = 47) 75.6 54.3–110.5 69.4 48.2–103.5 23.6 229.5 to 21.5 0.905 0.409
Placebo (n = 48) 79.3 54.6–122.0 82.5 60.2–106.0 211.9 236.4 to 19.2 0.246
Lipids
Total cholesterol, mg/dL
Vitamin D (n = 40) 180 158–201 190 166–217 3 211 to 11 0.419 0.168
Placebo (n = 37) 183 157–215 195 165–220 1 219 to 6.5 0.950
HDL-C, mg/dL
Vitamin D (n = 40) 59 42–68 64 51–71 20.5 25 to 4 0.937 0.769
Placebo (n = 39) 54 42–65 57 51–71 21 25 to 5 0.659
LDL-C, mg/dL
Vitamin D (n = 39) 100 80–124 109 87–132 1 212 to 12 0.120 0.096
Placebo (n = 37) 107 74–122 112 86–133 26 218 to 8 0.700
Triglycerides, mg/dL
Vitamin D (n = 39) 71 50–110 86 70–116 4 221 to 16 0.861 0.902
Placebo (n = 37) 86 48–125 103 65–145 21 217.5 to 23.5 0.904
Body composition
Fat mass, kg
Vitamin D (n = 48) 18.7 14.4–22.6 19.1 13.9–23.0 0 20.7 to 1.0 0.394 0.423
Placebo (n = 49) 22.3 16.3–25.5 22.5 16.0–25.0 0 20.6 to 0.7 0.693
Lean mass, kg
Vitamin D (n = 39) 58.6 55.7–61.2 59.1 55.8–61.3 3.3 25.7 to 9.2 0.478 0.155
Placebo (n = 37) 58.5 54.2–61.7 58.2 54.9–62.5 1.0 27.7 to 6.6 0.830

Abbreviations: HDL-C, high-density lipoprotein cholesterol; IQR, interquartile range; LDL-C, low-density lipoprotein cholesterol.
a
Changes between baseline and study end were calculated as the difference between the study end and baseline concentrations of biochemical
parameters.
b
Student t and Mann-Whitney U tests were used to compare the change in values between the vitamin D and placebo groups.
c
Paired Student t and Wilcoxon tests were used to compare biochemical characteristics at baseline and follow-up (within group differences).
4298 Lerchbaum et al Vitamin D and Testosterone in Men J Clin Endocrinol Metab, November 2017, 102(11):4292–4302

Table 3. Outcome Variables at Baseline and Visit 2, and Changes From Baseline to Visit 2
Baseline Visit Visit 2 Change From Baseline to Visit 2 Within-Group
Differences
Median IQR Median IQR Median IQR P Valuea P Valueb
TT, nmol/L
Vitamin D (n = 49) 18.7 15.9–21.9 18.2 15.9–20.6 20.1 21.6 to 2.9 0.902 0.214
Placebo (n = 49) 17.6 15.7–20.5 16.3 15–19.6 20.2 23.0 to 1.8 0.145
FT, ng/mL
Vitamin D (n = 48) 0.100 0.085–0.108 0.099 0.075–0.123 0 20.02 to 0.01 0.486 0.294
Placebo (n = 49) 0.096 0.079–0.110 0.092 0.074–0.118 0 20.02 to 0.02 0.840
SHBG, nmol/L

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Vitamin D (n = 48) 39.9 30.1–48.4 39.4 30–49.4 0.1 25.7 to 3.1 0.506 0.644
Placebo (n = 49) 39.7 31.2–54.1 38.7 29.4–49.5 21.4 210.1 to 3.8 0.129
FAI
Vitamin D (n = 48) 7.6 5.1–11.8 6.6 5.0–10.9 20.58 21.76 to 0.94 0.862 0.157
Placebo (n = 49) 6.9 4.9–10.0 5.6 4.7–9.5 20.72 22.05 to 0.98 0.060
Abbreviation: IQR, interquartile range.
a
Changes between baseline and visit 2 were calculated as the difference between the visit 2 and baseline concentrations of biochemical parameters.
Student t and Mann-Whitney U tests were used to compare the change in values between the vitamin D and placebo groups.
b
Paired Student t and Wilcoxon tests were used to compare biochemical characteristics at baseline and follow-up (within group differences).

Discussion differences might be explained by different study de-

In this RCT, we found no significant effect of vitamin D
therapy on TT levels in men. Regarding our secondary
end points, we found a significant difference in changes
in the QUICKI, and a trend toward a significant change
in the Matsuda index between the vitamin D and pla-
cebo groups, suggesting a decrease in insulin sensitivity
in the vitamin D group. We observed no significant
treatment effect on the remaining secondary outcome
measures, including gonadal endocrine parameters,
insulin resistance, AUCglucose, AUCinsulin, fat and
lean mass, and sexual, physical, and psychological
symptoms.
Our results are in line with a previous post hoc
analysis by Heijboer et al. (20) demonstrating no effect
of vitamin D supplementation on serum TT concen-
trations in three independent intervention studies in-
cluding male patients with heart failure (study 1), male
nursing home residents (study 2), and male non-Western
immigrants in the Netherlands (study 3). Those studies
were designed to investigate vitamin D effects on the
renin-angiotensin-aldosterone system (study 1), effects
of different vitamin D doses (study 2), and vitamin D
effects on insulin sensitivity (study 3). Similarly, the
study by Jorde et al. (19) did not show a significant
vitamin D effect on testosterone concentrations in
pooled data from three vitamin D RCTs performed in
Tromsø with weight reduction, insulin sensitivity,
and depression scores as end points. In contrast, we
previously found a significant increase in androgen
levels after vitamin D supplementation in obese men
undergoing a weight reduction program (18). Those
signs and subjects. Although the Pilz et al. study (18)
and the current study used similar doses (3,332 IU/d vs
2,857 IU/d, respectively), the Pilz et al. study (18) in-
vestigated vitamin D effects after 1 year of vitamin D
supplementation (vs 12 weeks in the Graz Vitamin
D&TT-RCT). Further, Pilz et al. (18) included subjects
with 25(OH)D levels ,50 nmol/L whereas the Graz
Vitamin D&TT-RCT investigated men with 25(OH)D
levels ,75 nmol/L. Other differences are related to the
study participants (significant weight loss in obese
subjects vs healthy men), baseline TT levels (11.4 nmol/L
vs 19.1 nmol/L) and the method used for TT mea-
surements (immunoassay vs mass spectrometry) for the
Pilz et al. study (18) and the current Graz Vitamin
D&TT-RCT report, respectively. Furthermore, Canguven
et al. (25) conducted an uncontrolled trial includ-
ing 102 male patients with 25(OH)D levels ,75 nmol/L
and observed a significant increase in TT levels after
vitamin D treatment. Those contradictory results might
be explained by different study duration (1 year vs
12 weeks) as well as by different dosing regimens (initial
vitamin D bolus after a vitamin D treatment regimen vs
weekly doses). Of note, because the Canguven et al.
study is an uncontrolled trial, no definite statement
regarding a real treatment effect of vitamin D can
be made.
Testosterone is an anabolic hormone with a wide
range of beneficial effects on men’s health, including
important physiological effects on brain, muscle, bone,
and fat mass (26). There is accumulating evidence sug-
gesting that androgen deficiency may contribute to the
onset and progression of cardiovascular disease and play
doi: 10.1210/jc.2017-01428 https://academic.oup.com/jcem 4299

context, Blomberg Jensen et al. (31)

observed a significant expression of
the VDR and vitamin D metabolizing
enzymes in the male reproductive
tract, including Leydig cells of the
testis. These data have raised the
question of whether vitamin D can
influence male reproductive hor-
mone production. The existence of
such an effect is supported by pre-

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vious studies suggesting that vitamin
D deficiency may contribute to re-
duced fertility and hypogonadism (11,
32). High LH and FSH levels in male
VDR knockout mice indicate the
presence of hypergonadotropic hypo-
gonadism (11). Recently, it has been
shown that vitamin D treatment
upregulates certain testis-specific genes
in mice (33), including ABCA1
(adenosine triphosphate-binding cas-
sette transporter 1). ABCA1 knockout
mice have significantly reduced intra-
testicular testosterone levels as well as
reduced sperm counts compared with
wild-type animals (34). Furthermore,
vitamin D significantly increased tes-
tosterone production in a human pri-
mary testicular cell culture model (35).
After 1,25-dihydroxyvitamin D sup-
plementation, 63 genes were signifi-
cantly upregulated in human testicular
Figure 2. Baseline and study end characteristics of study participants. Data are show as
cells, such as IGF-1, ALPL, DPP4, and
median with interquartile range. (a) QUICKI at study end and baseline. Mann-Whitney U test
was used to compare the vitamin D (n = 47) and placebo (n = 48) groups. (b) Changes genes associated with bone and the
between baseline and study end were calculated as the difference between the Matsuda immune system (34). Despite this
index at study end and baseline. Mann-Whitney U test was used to compare vitamin D promising evidence, we did not find at
(n = 45) and placebo (n = 48) groups.
significant vitamin D effect on andro-
gen levels in men in this RCT. This
an important role in the development of the metabolic suggests that previously observed associations between
syndrome in men (27). Thus, a causal relationship be- 25(OH)D and testosterone levels may be a consequence
tween vitamin D and testosterone, with a potential in- of confounding or reverse causation.
crease of testosterone levels after vitamin D treatment is Regarding secondary outcomes, we observed a sig-
of clinical interest. nificant difference in changes in the QUICKI between the
Several lines of evidence suggest a complex in- vitamin D and placebo groups as well as a trend toward a
terplay of vitamin D and androgen metabolism. In- significant difference in changes in the Matsuda index.
terestingly, it has been shown that androgens increase This finding is unexpected because previous observa-
1-a-hydroxylase, a key enzyme in vitamin D metabolism tional studies demonstrated a positive association of vi-
that converts 25(OH)D to 1,25-dihydroxyvitamin D tamin D and insulin sensitivity (4), whereas our results
(28). Furthermore, it has been demonstrated that the suggest a decrease of insulin sensitivity in the vitamin D
regulation of gene expression by vitamin D metabolites group. Evidence from previous RCTs revealed conflicting
is modified according to androgen levels (29). The VDR, results, however (33, 36). A positive vitamin D effect has
as well as key enzymes for vitamin D metabolism, are been demonstrated in insulin-resistant, vitamin D–deficient
widely expressed in human tissues and cells (30). In this women (33), whereas the study by Mousa et al. (36) did
4300 Lerchbaum et al Vitamin D and Testosterone in Men J Clin Endocrinol Metab, November 2017, 102(11):4292–4302

Table 4. Parameters of Mineral Metabolism at Baseline, Visit 2, and Study End, and Changes From Baseline
Change From Baseline Change From Baseline
Baseline Visit Visit 2 to Visit 2 Study End to Study End

Median IQR Median IQR Median IQR P Value Median IQR Median IQR P Value
25(OH)D, nmol/L
Vitamin D (n = 49) 52 42–65 82 76–98 32 21–44 ,0.001 107 89–119 53 36–65 ,0.001
Placebo (n = 49) 52 45–64 56 42–67 1 27 to 9 69 46–79 9 22 to 22
PTH, pg/mL
Vitamin D (n = 45) 48.2 37.0–54.4 45.0 33.9–53.2 21.3 26.6 to 8.2 0.009
Placebo (n = 48) 44.5 35.0–50.2 48.6 39.9–65.4 5.6 23.5 to 17.6
Serum calcium, mmol/L
Vitamin D (n = 45) 2.38 2.33–2.42 2.39 2.34–2.47 0.01 20.04 to 0.371 2.37 2.30–2.42 20.04 20.08 to 0.765

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0.09 0.04
Placebo (n = 48) 2.40 2.35–2.42 2.39 2.35–2.44 0.00 20.05 to 2.37 2.33–2.41 20.03 20.06 to
0.07 0.01
Urine calcium, mmol/L
Vitamin D (n = 45) 2.61 1.59–3.87 2.41 1.70–3.72 20.17 21.12 to 0.806
0.95
Placebo (n = 48) 2.49 1.33–4.00 2.44 1.07–4.42 20.25 21.05 to
1.38

Changes between baseline and visit 2, and baseline and study end were calculated as the difference between the study end (or visit 2) and baseline
concentrations of biochemical parameters. Student t and Mann-Whitney U tests were used to compare the change in values between the vitamin D and
placebo groups.
Abbreviation: IQR, interquartile range.

not demonstrate a significant vitamin D effect on insulin
sensitivity (determined via hyperinsulinemic-euglycemic
clamp) in vitamin D–deficient overweight or obese
adults. To our knowledge, there is no published RCT
demonstrating an adverse effect of vitamin D on insulin
sensitivity. Our finding, therefore, is difficult to explain
and we can only speculate on underlying mechanisms. It
should be noted, however, that study participants had
relatively high 25(OH)D levels at study end; there-
fore, our findings might be supported by previous in-
consistent results with possible U-shaped or nonlinear
associations that have been suggested for vitamin D and
cancer (5), cardiovascular disease (37), and mortality
(38, 39). Thus, we hypothesize that there might be a
U-shaped association of vitamin D status with insulin
sensitivity in this cohort of healthy men without insu-
lin resistance at baseline. Considering the previously
demonstrated positive effect of vitamin D treatment on
insulin sensitivity (35), our results might also be a chance
finding without an underlying true effect. Additional
large-scale RCTs addressing the effect of vitamin D on
insulin sensitivity in healthy subjects are warranted.
Our study has several limitations that should be noted.
First, we present data from a single-center study per-
formed in healthy middle-aged men; therefore, our results
may not be generalizable to other populations. Because
we investigated men with relatively high baseline TT
levels, we cannot exclude a vitamin D effect on testos-
terone levels in hypogonadal men or men with low-
normal TT levels. This issue is addressed in the second
arm of the Graz Vitamin D&TT-RCT, and results are
expected later in 2017. Furthermore, we investigated a
cohort of men with relatively high baseline 25(OH)D
levels. Thus, we cannot exclude vitamin D effects in men
with severe vitamin D deficiency. Given that a U-shaped
association of vitamin D levels with hypogonadism has
been observed in healthy middle-aged men (40), one
might speculate that an RCT aiming at target 25(OH)D
levels between 75 and 100 nmol/L would provide dif-
ferent results. Moreover, we cannot exclude that time
interval of vitamin D supplementation had an impact on
our study outcomes and that the use of daily instead of
weekly doses could have changed our results. Further-
more, due to the relatively short treatment period, we
cannot exclude substantial effects of vitamin D on an-
drogen levels with longer treatment. Moreover, we es-
timated insulin sensitivity using surrogate indices rather
than the gold standard method (i.e., hyperinsulinemic-
euglycemic clamp). Finally, we did not assess dietary
calcium intake.
Our study has also several strengths. First, this RCT
was specifically designed to investigate vitamin D effects
on TT concentrations in men. Second, we used state-of-
the-art and standardized methods to measure 25(OH)D
as well as TT concentrations in our samples (41). Further,
we included a relatively large number of participants and
the dropout rate was low. In addition, although we did not
use gold standard methods for evaluation of insulin sen-
sitivity, we analyzed data derived from OGTTs, allowing a
more precise estimation of insulin sensitivity and insulin
resistance than the simple use of fasting values.
In summary, we found no significant vitamin D effect
on TT concentrations in this cohort of middle-aged healthy
men with normal baseline TT levels, confirming the results
doi: 10.1210/jc.2017-01428
of previous preliminary RCTs, but we observed a sig-
nificant decrease in QUICKI. The latter finding points to
the need for additional studies investigating a potential
adverse effect of vitamin D on insulin sensitivity and
insulin resistance in large cohorts of healthy subjects.
Regarding vitamin D and TT, we recommend further
investigation of potential vitamin D effects on androgen
levels in different cohorts, including hypogonadal men
with severe vitamin D deficiency.
Acknowledgments
We thank all study participants, Roswitha Gumpold for re-
cruitment of patients, Cornelia Missbrenner and the members of
Endocrinology Laboratory platform for continuous support,
David Lerchbaum for scientific discussion and statistical support,
and Fresenius Kabi for providing the study medication.
Financial Support: The Graz Vitamin D&TT-RCT was
supported by funding from the Austrian National Bank (OeNB
Jubilaeumsfonds Project 14846).
Clinical Trial Information: EudraCT no. 2011-003575-11
(registered 3 May 2013); ClinicalTrials.gov no. NCT01748370
(registered 10 December 2012).
Correspondence and Reprint Requests: Elisabeth
Lerchbaum, MD, Medical University Graz, Department of
Internal Medicine, Division of Endocrinology and Diabetology,
Auenbruggerplatz 15, 8036 Graz, Austria. E-mail: elisabeth.
lerchbaum@medunigraz.at.
Disclosure Summary: The authors have nothing to
disclose.
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