Introduction: There are reports of a possible relationship between melatonin, a hormone secreted by the pineal gland, and exercise. Objective: The present study aims to investigate how diurnal and nocturnal strenuous exercise affects melatonin levels. Methods: The study enrolled 10 healthy sedentary males who did not actively exercise. The subjects had a mean age of 22.20±0.24 years, a mean height of 174.60±2.33 cm, and a mean weight of 69.70±2.42 kg. Two blood samples were collected from the subjects, one at rest, at 10: 00 am, and the other immediately after strenuous exercise. Likewise, blood samples were taken from the same group of subjects after 48 hours: at 24: 00 hours at rest and immediately after strenuous exercise. Samples were analyzed using the ELISA method to determine the serum melatonin levels (pg/ml). Results: By comparing the values at rest and after exercise, it was found that serum melatonin values remained unchanged with exercise. Serum melatonin values at rest or post-exercise measured at night were higher when compared with those measured during the day (p<0.05). Conclusions: Higher levels of melatonin found in the study appear to result from the increased release of melatonin at night, and not from exercise. The results of this study indicate that strenuous exercise carried out day or night, did not significantly influence serum melatonin levels.