Background:Night shift work has been associated with an increased risk for breast and prostate cancer. The effect of circadian disruption on sex steroid production is a possible underlying mechanism, underinvestigated in humans. We have assessed daily rhythms of sex hormones and melatonin in night and day shift workers of both sexes. Methods: We recruited 75 night and 42 day workers, aged 22-64 years, in different working settings. Participants collected urine samples from all voids over 24 hours on a working day. Urinary concentrations of 16 sex steroid hormones and metabolites (estrogens, progestagens and androgens) and 6-sulfatoxymelatonin were measured in all samples. Mean levels and peak time of total and individual metabolite production were compared between night and day workers. Results: Night workers had higher levels of total progestagens (geometric mean ratio (GMR) 1.65; 95% confidence interval (CI) 1.17, 2.32) and androgens (GMR: 1.44; 95% CI 1.03, 2.00), compared to day workers, after adjusting for potential confounders. The increased sex hormone levels among night shift workers were not related to the observed suppression of 6-sulfatoxymelatonin. Peak time of androgens was significantly later among night workers, compared to day workers (testosterone: 12:14 h; 10:06, 14:48 vs 08:35 h; 06:52, 10:46). Conclusions: We found increased levels of progestagens and androgens as well as delayed peak androgen production in night shift workers, compared to day workers. Impact: The increase and mistiming of sex hormone production may explain part of the increased risk for hormone-related cancers observed in night shift workers.