OBJECTIVE: To quantify how representative a single measure of reproductive hormone is of the daily hormonal profile using data from detailed hormonal sampling in the saline placebo-treated arm conducted over several hours. DESIGN: Retrospective analysis of data from previous interventional research studies evaluating reproductive hormones. SUBJECTS: Overall, 266 individuals including healthy men and women (n=142), and those with reproductive disorders/states (n=124) [11 with functional hypothalamic amenorrhoea (FHA), 6 with polycystic ovary syndrome (PCOS), 62 women and 32 men with hypoactive sexual desire disorder (HSDD), and 13 post-menopausal women] were included in the analysis. INTERVENTIONS: Data from 266 individuals who had undergone detailed hormonal sampling in the saline placebo-treated arms of previous research studies was used to quantify the variability in reproductive hormones due to pulsatile secretion, diurnal variation, and feeding using coefficient of variation (CV) and entropy. MAIN OUTCOME MEASURES: The ability of a single measure of reproductive hormone to quantify the variability in reproductive hormones due to pulsatile secretion, diurnal variation, and nutrient-intake. RESULTS: The initial morning value of reproductive hormones was typically higher than the mean value throughout the day (percentage decrease from initial morning measure to daily mean: LH 18.4%, FSH 9.7%, testosterone 9.2%, and estradiol 2.1%). LH was the most variable (CV 28%), followed by sex-steroids (testosterone 12%, estradiol 13%), whereas FSH was the least variable reproductive hormone (CV 8%). In healthy men, testosterone fell between 9am and 5pm by 14.9% (95% CI 4.2, 25.5%), although morning levels correlated with (and could be predicted from) late afternoon levels in the same individual (r2=0.53, p<0.0001). Testosterone was reduced more after a mixed meal (by 34.3%) than during ad libitum feeding (9.5%), or after an oral glucose load (6.0%), or an intravenous glucose load (7.4%). CONCLUSION: Quantification of the variability of a single measure of reproductive hormones informs the reliability of reproductive hormone assessment.