Background  Estimates of intraindividual variation in hormone levels provide the basis for interpreting hormone measurements clinically and for developing eligibility criteria for trials of hormone replacement therapy. However, reliable systematic estimates of such variation are lacking.

Objective  To estimate intraindividual variation of serum total, free and bioavailable testosterone (T), dihydrotestosterone (DHT), SHBG, LH, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), oestrone, oestradiol and cortisol, and the contributions of biological and assay variation to the total.

Design  Paired blood samples were obtained 1–3 days apart at entry and again 3 months and 6 months later (maximum six samples per subject). Each sample consisted of a pool of equal aliquots of two blood draws 20 min apart.

Study participants  Men aged 30–79 years were randomly selected from the respondents to the Boston Area Community Health Survey, a study of the health of the general population of Boston, MA, USA. Analysis was based on 132 men, including 121 who completed all six visits, 8 who completed the first two visits and 3 who completed the first four visits.

Measurements  Day-to-day and 3-month (long-term) intraindividual standard deviations, after transforming measurements to logarithms to eliminate the contribution of hormone level to intraindividual variation.

Results  Biological variation generally accounted for more of total intraindividual variation than did assay variation. Day-to-day biological variation accounted for more of the total than did long-term biological variation. Short-term variability was greater in hormones with pulsatile secretion (e.g. LH) than those that exhibit less ultradian variation. Depending on the hormone, the intraindividual standard deviations imply that a clinician can expect to see a difference exceeding 18–28% about half the time when two measurements are made on a subject. The difference will exceed 27–54% about a quarter of the time.

Conclusions  Given the level of intraindividual variability in hormone levels found in this study, one sample is generally not sufficient to characterize an individual's hormone levels but collecting more than three is probably not warranted. This is true for clinical measurements and for hormone measurements used to determine eligibility for a clinical trial of hormone replacement therapy.