Abstract Background: Women with breast cancer have decreased levels of melatonin or its metabolite in plasma and/or urine. Methods: We measured serum melatonin, urinary 6-sulfatoxymelatonin, catecholamines, and cortisol in 141 sedentary young female participants in a clinical trial comparing 150 min/wk aerobic exercise for 4 months to no-exercise controls. Demographics, health surveys, body composition, sleep quality, fitness levels, and blood and urine samples were obtained at baseline and 16 weeks. Results: There were no differences between groups at baseline in demographics, exercise, sleep habits, or study hormones. There were also no significant differences between groups in any of the hormones at 16 weeks. Conclusion: Sixteen weeks of exercise had minimal effects on melatonin secretion of young women. Impact: There is convincing evidence that exercise protects against breast cancer, but this does not appear to occur through changes in melatonin secretion. Cancer Epidemiol Biomarkers Prev; 22(9); 1634–6. ©2013 AACR.