Abstract STUDY QUESTION How do the calciotropic hormones (25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and intact parathyroid hormone (iPTH)) vary across the menstrual cycle and do cyclic patterns of reproductive hormones (estradiol, progesterone, LH, FSH) differ by vitamin D status? SUMMARY ANSWER Calciotropic hormones vary minimally across the menstrual cycle; however, women with 25-hydroxyvitamin D below 30 ng/ml have lower mean estradiol across the menstrual cycle. WHAT IS KNOWN ALREADY Prior human studies suggest that vitamin D status is associated with fecundability, but the mechanism is unknown. Exogenous estrogens and prolonged changes in endogenous estradiol (pregnancy or menopause) influence concentrations of 25-hydroxyvitamin D. In vitro, treatment with 1,25-dihydroxyvitamin D increases steroidogenesis in ovarian granulosa cells. There are little data about changes in calciotropic hormones across the menstrual cycle or cyclic patterns of reproductive hormones by categories of vitamin D status. STUDY DESIGN, SIZE, DURATION A prospective cohort study of 89 self-identified white women aged 18–44, across two menstrual cycles. Participants were a subset of the BioCycle Study, a community-based study conducted at the University of Buffalo, 2005–2007. PARTICIPANTS/MATERIALS, SETTING, METHODS Eligible participants had self-reported regular menstrual cycles between 21 and 35 days and were not using hormonal contraception or vitamins. Early morning fasting blood samples were drawn at up to eight study visits per cycle. Visits were timed to capture information in all cycle phases. Serum samples for 89 women (N = 163 menstrual cycles) were analyzed for estradiol, progesterone, LH, FSH and 25-hydroxyvitamin D (25(OH)D). Variability in calciotropic hormones within and across menstrual cycles was assessed using intraclass correlation coefficients and non-linear mixed models. Given the relative stability of the calciotropic hormones across the menstrual cycle, non-linear mixed models were used to examine differences in the cyclic patterns of estradiol, progesterone, LH and FSH by categories of each calciotropic hormone (split at the median). These models were conducted for all ovulatory cycles (N = 142 ovulatory menstrual cycles) and were adjusted for age, BMI (measured in clinic) and self-reported physical activity. MAIN RESULTS AND THE ROLE OF CHANCE Median 25(OH)D concentration was 29.5 ng/ml (SD 8.4), and only 6% of women had vitamin D deficiency (<20 ng/ml). The mean concentration of 25(OH)D did not differ between the luteal and follicular phase; however, both 1,25(OH)2D and iPTH showed small fluctuations across the menstrual cycle with the highest 1,25(OH)2D (and lowest iPTH) in the luteal phase. Compared with women who had mean 25(OH)D ≥30 ng/ml, women with lower 25(OH)D had 13.8% lower mean estradiol (95% confidence interval: −22.0, −4.7) and 10.8% lower free estradiol (95% CI: −0.07, −0.004). Additionally, compared to women with iPTH ≤36 pg/ml, women with higher concentrations of iPTH had 12.7% lower mean estradiol (95% CI: −18.7, −6.3) and 7.3% lower progesterone (95% CI: −13.3, −0.9). No differences in the cyclic pattern of any of the reproductive hormones were observed comparing cycles with higher and lower 1,25(OH)2D. LIMITATIONS, REASONS FOR CAUTION Women included in this study had self-reported ‘regular’ menstrual cycles and very few were found to have 25(OH)D deficiency. This limits our ability to examine cycle characteristics, anovulation and the effects of concentrations of the calciotropic hormones found in deficient individuals. Additionally, the results may not be generalizable to women with irregular cycles, other races, or populations with a higher prevalence of vitamin D deficiency. WIDER IMPLICATIONS OF THE FINDINGS These findings support current clinical practice that does not time testing for vitamin D deficiency to the menstrual cycle phase. We find that women with lower vitamin D status (lower 25(OH)D or higher iPTH) have lower mean concentrations of estradiol across the menstrual cycle. Although this study cannot identify a mechanism of action, further in vitro work or clinical trials may help elucidate the biologic mechanisms linking calciotropic and reproductive hormones. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Intramural Research Programs of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (contract numbers: HHSN275200403394C, HHSN275201100002I and Task 1 HHSN27500001) and the National Institute of Environmental Health Sciences. There are no competing interests.