The purpose of this study was to systematically evaluate the effect of endurance exercise duration on hormone concentrations in male subjects while controlling for exercise intensity and training status. Eight endurance-trained males (19-49 years) completed a resting control session and three treadmill runs of 40, 80, and 120 min at 55% of VO2max . Blood samples were drawn before the session and then 1, 2, 3 and 4 h after the start of the run. Plasma was analyzed for luteinizing hormone (LH), dehydroepiandrosterone sulfate (DHEAS), cortisol, and free and total testosterone. LH was significantly greater at rest compared to the running sessions. Both free and total testosterone generally increased in the first hour of the 80 and 120 min runs and then showed a trend for a steady decline for the next 3 h of recovery. Dehydroepiandrosterone sulfate increased in a dose-response manner with the greatest increases observed during the 120-min run, followed by the 80-min run. Cortisol only increased in response to the 120-min run and showed a decline across time in all other sessions. The ratios of anabolic hormones (testosterone and DHEAS) to cortisol were greater during the resting session and the 40-min run compared to the longer runs. The results indicate that exercise duration has independent effects on the hormonal response to endurance exercise. At a low intensity, longer duration runs are necessary to stimulate increased levels of testosterone, DHEAS and cortisol and beyond 80 min of running there is a shift to a more catabolic hormonal environment.