Some studies suggest that exogenous melatonin supplementation may improve athletic performance in hot humid environments because of its precooling effect. However, melatonin is also consumed as a sleep aid for its depressive effects on the central nervous system, which may hinder performance. Therefore, this study was conducted to determine whether consuming a 5 mg supplement of melatonin would affect performance in a laboratory-simulated 32.2 kilometer cycling time trial. The time trial was conducted in a thermoneutral environment to separate CNS depressive effects of the melatonin from the cooling effects. Trained male subjects (n = 10; VO2 max = 62.7 ± 6.3 ml/kg/min; age = 25.1 ± 4.0 yr; mass = 69.9 ± 9.1 kg; height = 176.0 ± 7.1 cm), performed three 32.2 kilometer time trials on an electronically braked cycle ergometer. The first trial was a familiarization. During the two experimental trials subjects received in a random order either a placebo or a 5 mg melatonin supplement 15 min prior to exercise in a double-blind, crossover design. Variables were measured before exercise and at 8 kilometer intervals. Mean 32.2 kilometer time trial completion times for the melatonin (64.94 ± 5.95 min) and placebo (65.26 ± 6.85 min) trials were not different (P = 0.682). Mean time trial power output for the melatonin (190.4 ± 40.4 watts) and placebo (190.0 ± 45.7 watts) trials were not different (P = 0.927). Rectal temperature was not significantly different for melatonin compared with placebo (P = 0.827). These results suggest that a 5 mg melatonin supplement administered 15 min prior to exercise does not measurably impact performance of a 32.2 kilometer cycling time trial in a thermoneutral environment.