Dihydrotestosterone (DHT) exerts both functional and signaling effects extending beyond the effects of testosterone in rodent skeletal muscle. As a primer for investigating the role of DHT in human skeletal muscle function, this study aimed to determine whether circulating DHT is acutely elevated in men following a bout of repeat sprint exercise and to establish the importance of training status and sprint performance to this response. Fourteen healthy active young men (VO2max 61.0 ± 8.1 ml.kg BM-1.min-1) performed a bout of repeat sprint cycle exercise at a target workload based on an incremental work-rate maximum (10x30s @ 150% Wmax with 90-s recovery). Venous blood samples were collected pre-, 5 minutes post- and 60 minutes post-exercise. 5 minutes post-exercise there were significant elevations in total testosterone (TT; P<0.001), free testosterone (FT; P<0.001) and DHT (P=0.004), which returned to baseline after one hour. Changes in DHT with exercise (5' Post-Pre) correlated significantly with changes in TT (r=0.870; P<0.001) and FT (r=0.914; P<0.001). Sprinting cadence correlated with changes in FT (r=0.697; P=0.006), DHT (r=0.625; P=0.017) and TT (r=0.603; P=0.022) and habitual training volume correlated with the change in TT (r=0.569, P=0.034). In conclusion, our data demonstrates that DHT is acutely elevated following sprint cycle exercise and that this response is influenced by cycling cadence. The importance of DHT in the context of exercise training and sports performance remains to be determined.