We evaluated the effects of a higher dose of vitamin D supplementation (2500 IU daily) than used in the Women's Health Initiative on several vascular parameters and markers of CVD risk. In our study, treated subjects had circulating 25(OH)D levels that increased, on average, by over 15 ng/mL and over 90% of treated subjects achieved 25(OH)D levels >30 ng/mL. Nevertheless, we did not observe an improvement in FMD or arterial stiffness measures. Similarly, we did not observe differential improvements in central or peripheral blood pressures or CRP levels between treatment groups.
Our prospective, randomized, blinded study findings did not show improvements in FMD or either arterial stiffness measure after treatment with VitD. The few randomized controlled trials that have evaluated the effects of vitamin D supplementation on endothelial function or arterial stiffness have had mixed results in regard to FMD 
, PWV 
, and AIx 
; however, those studies were notably smaller than our study and predominantly were performed in adults with medical conditions such as kidney disease and/or type II diabetes mellitus. Similarly, there are mixed reports regarding changes in blood pressure following VitD supplementation 
. Despite higher blood pressures in individuals with lower 25(OH)D levels, we found no change in blood pressure after VitD supplementation, in agreement with the largest of these trials 
. Despite higher CRP levels in individuals with lower 25(OH)D levels, we also found no change in CRP after supplementation, a finding consistent with previous reports 
. The lack of an effect of VitD supplementation on these markers of CVD risk was confirmed in our subgroup analyses restricted to participants with low VitD status at baseline using multivariable linear models which showed that treatment group did not influence change in FMD, PWV, or AIx. Although individuals with low VitD status at baseline had a small increase in maximum relative FMD after 4 months, this increase was not influenced by treatment group. Our results challenge the hypothesis that VitD supplementation reduces CVD risk.
Vitamin D receptors are widely distributed throughout the body and have been isolated from vascular endothelial cells and cardiac myocytes 
. Mechanistically it is conceivable that low VitD levels could have deleterious CVD effects by dysregulation of systemic calcium metabolism, as cardiac myocyte contraction depends on calcium homeostasis and since coronary artery calcification is predictive of CVD risk 
. Also, VitD modulates lymphocytic cytokine production 
, potentially affecting growth and proliferation of vascular smooth muscle cells and cardiomyocytes 
, stimulating vascular tissue anticoagulant activity 
, and suppressing renin gene expression 
, which could lead to the clinical manifestations of hypertension, coronary artery disease, and congestive heart failure. However, mechanistic hypotheses do not necessarily imply that supplementing individuals with low 25(OH)D levels will reverse these adverse processes and reduce CVD risk 
. Blood pressure reductions have been observed with VitD supplementation 
, but the majority of studies have shown no change 
. Furthermore, 2 randomized controlled trials showed no improvement in left ventricular function in patients with heart failure randomized to VitD therapy 
Although plausible biological mechanisms and epidemiological data suggest that VitD deficiency may increase and VitD supplementation may reduce CVD risk, observational studies cannot account for unmeasured confounders. In our study, as in others, individuals with low VitD tended to have a greater CVD risk factor burden 
. It has been proposed that low VitD is responsible for these observations; however, it is possible that these CVD risk factors simply are associated with lower 25(OH)D levels. Indeed, previous studies have shown that the risk factors for VitD deficiency are similar to traditional CVD risk factors 
. In our study, despite an average increase in circulating 25(OH)D levels of nearly 16 ng/mL, we observed no improvements in FMD, PWV or AIx, surrogate CVD risk markers that predict initial and recurrent CVD events 
Our endpoints are surrogate markers for CVD risk; we did not evaluate CVD death, myocardial infarction, or stroke. Although our markers are predictive of CVD events and are well-established research tools, they are imperfect and an absence of change in these measures does not exclude the possibility that VitD supplementation may reduce CVD risk 
. Participants in this study were generally healthy post-menopausal women with typical 25(OH)D levels. It is possible that VitD supplementation in men or in individuals with higher baseline CVD risk or certain co-morbidities, including abnormal endothelial function or lower 25(OH)D levels, may reduce CVD risk. Additionally, research on the effects of VitD supplementation is challenged by the absence of a widely accepted definition of vitamin D “insufficiency.” Proposed “reasonable” 25(OH)D levels range from 20 ng/mL (50 nmol/L) to 32 ng/mL (80 nmol/L) 
and some advocate VitD supplementation in individuals with 25(OH)D levels that are <50 ng/mL 
. Subjects in this study started with a wide range of 25(OH)D levels; those subjects with higher starting levels may have had a blunted arterial response to treatment despite potentially being randomized to the treatment group. Similarly, there is considerable debate regarding the optimal dose for VitD supplementation and repletion in adults. Expert opinion for supplemental dosing ranges from 600–1000 IU (20–25 µg) to 2000 IU (50 µg) daily 
, although some have argued that even the latter dose may be inadequate and higher doses frequently are used clinically 
. Given the inconsistencies in the literature, we cannot exclude the possibility that supplementation with even higher doses of VitD for a longer duration would show a benefit in terms of CVD risk; however, interventions that reduce CVD risk also tend to improve endothelial function and arterial stiffness soon after initiating treatment, suggesting that 4 months was a reasonable duration of treatment 
. Based on revised estimates using a mixed effects general linear model and restricted maximum likelihood estimation, with 55 subjects per arm we had over 90% power to detect a 1.5% difference in FMD. For PWV, we observed essentially no change PWV and the SDs were very small, however there still is a small chance that we missed a true difference between groups.
In the largest prospective randomized clinical trial of VitD supplementation that used a dose of VitD that normalized VitD levels in most participants, VitD supplementation did not improve endothelial function, arterial stiffness, reduce CRP or improve blood pressure in healthy, post-menopausal women. This study does not support the use of VitD supplementation to reduce CVD risk; however, long-term outcomes studies of this intervention are needed.