AbstractBACKGROUNDMelatonin is synthesized in greater concentration at night, and it plays an important role in sleep regulation. This study aimed to evaluate the melatonin concentration in milk collected by milking during the night and evaluates its effect, with or without tryptophan supplementation, in the sleep quality of adult Wistar rats.RESULTSA difference (P < 0.05) was observed between the milking times, where the milk obtained at 02:00 presented a higher melatonin concentration (39.43 pg mL−1) than that acquired at 15:00 (4.03 pg mL−1). A biological assay was also performed on 32 male adult Wistar rats distributed among four groups (n = 8): those receiving an AIN‐93M diet (control group) and three test groups [diets containing milk from milking at 02:00 (M2h), milking at 15:00 (M15h), and milking at 02:00 plus tryptophan supplementation (M2hT)] for 28 days. It was observed that the control group did not differ (P > 0.05) from the M15h group in terms of the levels of blood melatonin and urinary sulfatoxymelatonin, but differed from groups M2h and M2hT, whereas group M2hT presented higher blood melatonin and urinary sulfatoxymelatonin concentrations.CONCLUSIONCombining the techniques of night milking with tryptophan supplementation resulted in production of milk that improves sleep quality in rats. © 2013 Society of Chemical Industry