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OBJECTIVE: Data from real world settings on circadian disruption and subsequent hormone-related changes may explain the higher risk of hormone-dependent cancers among night shift workers.The present study examines the melatonin and sex steroid hormone levels among night shift workers. METHODS: We included 44 male, rotating shift workers from a car factory in Spain, sampled both at the end of a 3-week night shift (22:00–06:00 hrs) and a 3-week early morning shift (06:00–14:00 hrs). Participants collected all urine voids over 24-hours during each shift. Urinary concentrations of sex steroid hormones (estrogens, androgens and progestogens) and 6-sulfatoxymelatonin (aMT6s, major melatonin metabolite) were determined. Individual cosinor analysis was used to derive the acrophase (peak time) and area under the curve (total production). Linear mixed models examined intraindividual associations between night shift work and log-transformed 24-hour peak time and total production of hormones compared to early morning shift work. RESULTS: The acrophase was delayed during the night shift for aMT6s [geometric mean difference (GMD) 7.53 hrs, 95% confidence interval (CI) 4.46–10.60], androgens (eg, testosterone: GMD 6.83 hrs, 95% CI 0.34–13.32) and progestogens (eg, 17-hydroxyprogesterone: GMD 4.54 hrs, 95% CI 2.92–6.16) compared to the early morning shift. We found a higher production of adrenal androgen 11-oxoandrosterone/11-oxoetiocholanolone [geometric mean ratio (GMR) 1.43, 95% CI 1.12–1.81], and a lower production of adrenal progestogen 16-cysteinylprogesterone (GMR 0.79, 95% CI 0.67–0.93) during the night shift compared to the early morning shift levels. CONCLUSIONS: Night shift work was associated with melatonin and sex hormone-related changes in timing and total production, providing insight into the mechanistic path for its association with hormone-dependent cancer.