The aim of the present prospective longitudinal study was to investigate the hormonal response in overtrained athletes at rest and during exercise consisting of a short-term exhaustive endurance test on a cycle ergometer at an intensity 10% above the individual anaerobic threshold. Over a period of 19± 1 months, 17 male endurance athletes (cyclists and triathletes; age 23.4 ± 1.6 yr; ˙VO2max 61.2 ± 1.8 mL·min-1·kg-1; means ± SEM) were examined five times on two separate days under standardized conditions. Short-term overtraining states (OT, N = 15) were primarily induced by an increase of frequency of high-intensive bouts of exercise or competitions without increase of the total amount of training. OT was compared with normal training states intraindividually (NS, N = 62). During OT, the time to exhaustion of the exercise test was significantly decreased by 27% on average. At rest and during exercise, the concentrations in plasma and the nocturnal excretion in urine of free epinephrine and norepinephrine were not significantly changed during OT. At physical rest, the concentrations of(free) testosterone, cortisol, luteinizing hormone, follicle-stimulating hormone, adrenocorticotropic hormone, growth hormone, and insulin during OT were comparable with those during NS. A significantly (P < 0.025) lower maximal exercise-induced increase of the adrenocorticotropic hormone and growth hormone, as well as a trend for a decrease of cortisol (P = 0.060) and insulin (P = 0.036), was measured. The response of free catecholamines as well as the ergometric performance of an all-out 30-s test was unchanged. Serum urea, uric acid, ferritin, and activity of creatine kinase showed no differences between conditions. In conclusion, the results confirm the hypothesis of a hypothalamo-pituitary dysregulation during OT expressed by an impaired response of pituitary hormones to exhaustive short-endurance exercise.