In this RCT among men with low serum TT concentrations at baseline, we found no significant effect of vitamin D treatment on serum TT levels or secondary end points. When analyses were restricted to men with 25(OH)D levels < 50 nmol/l, we found a significant increase of SHBG levels after 12 weeks in the placebo group, whereas SHBG levels remained unchanged in the vitamin D group. There was no significant effect on serum TT levels or the remaining secondary outcome parameters in this subgroup.

Our results demonstrating no significant effect on TT concentrations are in line with our previous data from men with normal serum TT concentrations at baseline [17]. Correspondingly, a previous post hoc analysis by Heijboer et al. [21] did not find a significant treatment effect. Heijboer et al. [21] investigated vitamin D effects on TT concentrations in three independent studies involving men with heart failure, male nursing home residents as well as male non-Western immigrants in the Netherlands. In addition, Jorde et al. [22] did not find a significant vitamin D effect on TT concentrations in pooled data from three vitamin D RCTs performed in Tromsö with weight reduction, insulin sensitivity, and depression scores as end points. Recently, Zitterman et al. [23] performed a pre-specified secondary analysis of the EVITA (effect of vitamin D on mortality in heart failure) RCT. The authors analyzed the effect of a daily vitamin D supplement of 4000 IU for 3 years (n = 71) vs. placebo (n = 62) on TT, SHBG, FT, and bioactive T (BAT) in men with 25(OH)D concentrations < 75 nmol/l. At study end, there was no between-group difference regarding androgen levels and SHBG. Consistently, our findings suggest that previous associations between 25(OH)D and testosterone status may have been rather the consequence of confounding and/or reverse causation than of a causal effect of vitamin D on testosterone status.

Interestingly, in men with 25(OH)D levels < 50 nmol/l at baseline, we observed a significant treatment effect on SHBG, the major carrier protein of testosterone, after 12 weeks of vitamin D supplementation. In detail, SHBG levels remained similar in the vitamin D group, whereas SHBG levels increased in the placebo group. Our findings are difficult to interpret and we cannot exclude that they are caused by chance. As the change in SHBG levels was not accompanied by a change in serum TT or FT levels, the clinical relevance of our finding remains to be determined. Data from men with normal serum TT levels participating the Graz Vitamin D&TT-RCT suggest a significant decrease of SHBG levels after 3 months in the vitamin D group but not in the placebo group [17]. It should, however, be emphasized that no significant treatment effect was found in eugonadal men [17] and when analyzing an RCT it is the comparison between the two treatment arms, that is of interest. Correspondingly, Zittermann et al. [23] observed no treatment effect on SHBG levels in the EVITA trial. Those different results might be explained by different study duration (3 months vs. 3 years), study participants (healthy men vs. men with advanced heart failure) and vitamin D dosing regimens (20,000 IU/week vs. 4000 IU/day) used in the studies. In this context, it should also be noted that previous observational studies found an inverse association of 25(OH)D and SHBG levels [16, 24].

Our results regarding varying serum TT levels assessed via immunoassay and ID-LC–MS/MS supports previous statements on the unreliable results of the currently used immunoassays [25] as well as the need for at least two measurements of low TT levels to confirm the diagnosis of hypogonadism [26]. Due to feasibility reasons, we used TT levels measured once by immunoassay to get immediate results, as MS was not available for these measurements at our department. Nevertheless, in light of the large discrepancies between baseline serum TT levels assessed by different methods (12.7 nmol/l vs. 8.2 nmol/l for ID-LC–MS/MS and immunoassay, respectively), the use of ID-LC–MS/MS for measuring TT levels should be considered for evaluation of inclusion criteria in future studies.

Our results in men with normal serum TT levels suggest an adverse effect of vitamin D supplementation on insulin sensitivity [17]. In contrast, in men with low serum TT levels at baseline we found no significant effect on metabolic parameters including insulin sensitivity. Previous evidence on vitamin D and insulin sensitivity is inconsistent. Observational studies point towards a positive association of vitamin D and insulin sensitivity [2]. Evidence from previous RCTs revealed, however, conflicting results [27, 28]. A positive vitamin D effect has been demonstrated in insulin-resistant, vitamin D-deficient women [28]. Mousa et al. [27] failed to demonstrate a significant vitamin D effect on insulin sensitivity (determined via hyperinsulinemic/euglycemic clamp) in vitamin D-deficient overweight or obese adults, which is in line with our results. We, therefore, cannot exclude that our previous findings [17] were caused by chance.

Our study has several limitations that should be noted. First, we used TT measured by immunoassay for evaluation of inclusion criteria. As TT levels measured by ID-LC–MS/MS were higher than expected, we cannot exclude significant vitamin D effects on androgen levels in men with lower TT levels at baseline. Further, as we investigated a cohort of men with relatively high baseline 25(OH)D levels, we cannot exclude vitamin D effects in men with severe vitamin D deficiency. This notion is supported by the fact that subgroup analyses of men with 25(OH)D levels < 50 nmol/l revealed a significant effect on SHBG levels at study end. Given that a U-shaped association of vitamin D levels with hypogonadism has been observed previously, one might speculate that a RCT aiming at target 25(OH)D levels between 75 and 100 nmol/l would provide different results. Further, time interval of vitamin D supplementation (daily instead of weekly doses) as well as the relatively short treatment period might have had an impact on our study outcome. We cannot exclude substantial effects of vitamin D on androgen levels with different doses, time intervals or longer treatment. As we present results from a single-center study performed in healthy middle-aged men with low serum TT levels, our results may not be generalizable to other populations.

Strengths of our study include the study design of an RCT as well as the use of state-of-the-art and standardized methods to measure 25(OH)D as well as TT concentrations in our samples [26]. Further, we included a relatively large number of participants and the dropout rate was low.

In summary, we found no significant vitamin D effect on androgen levels including TT, FT and FAI concentrations in this cohort of middle-aged healthy men with low baseline serum TT levels. This finding confirms our previous results in men with normal serum TT levels and suggests that vitamin D treatment has no clinical relevant effect on testosterone levels in men. Of note, future studies should only be performed in truly vitamin D-deficient subjects (< 25 or 30 nmol/l) and low testosterone levels to evaluate vitamin D effects on testosterone levels.