Brandenberger, KJ, Ingalls, CP, Rupp, JC, and Doyle, JA. Consumption of a 5-mg melatonin supplement does not affect 32.2-km cycling time trial performance. J Strength Cond Res 32(10): 2872–2877, 2018—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 (CNS), 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-km 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; V[Combining Dot Above]O2max = 62.7 ± 6.3 ml·kg−1·min−1; age = 25.1 ± 4.0 years; mass = 69.9 ± 9.1 kg; height = 176.0 ± 7.1 cm) performed three 32.2-km time trials on an electronically braked cycle ergometer. The first trial was a familiarization. During the 2 experimental trials, subjects received in a random order either a placebo or a 5-mg melatonin supplement 15 minutes before exercise in a double-blind, crossover design. Variables were measured before exercise and at 8-km intervals. The mean 32.2-km time trial completion times for the melatonin (64.94 ± 5.95 minutes) and placebo (65.26 ± 6.85 minutes) trials were not different (p = 0.682). The mean time trial power output for the melatonin (190.4 ± 40.4 watts) and placebo (190.0 ± 45.7 watts) trials was 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 minutes before exercise does not measurably impact the performance of a 32.2-km cycling time trial in a thermoneutral environment.