This study was designed to compare a first bout of high-intensity endurance exercise with a second bout of similar exercise on the same day, and thereby test the hypothesis that the endocrine response elicited by a second bout is more pronounced compared with a single bout of exercise. Nine male, elite endurance athletes participated in three trials of 24-h duration: 1) complete bed rest (REST), 2) one bout of exercise (ONE), and 3) two bouts of exercise separated by a 3-h rest period (TWO). Each exercise bout consisted of a 10-min warm-up at 50% of VO(2max) followed by 65 min at 75% of VO(2max) on a cycle ergometer. Exercise was performed between 11:00 a.m. and 12:15 a.m. (only in TWO) and 3:15 and 4:30 p.m. (both ONE and TWO). The subjects rested in bed at all hours except when exercising. Blood was sampled 11 times at identical time-points until 7:30 a.m. the next morning. We observed significantly increased levels of epinephrine, norepinephrine, ACTH, cortisol, and growth hormone, and decreased levels of testosterone during and/or after the second bout of exercise compared with the first bout. No difference was observed for insulin, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, free fraction of thyroxin or insulin-like growth factor 1. Thus, this study demonstrates a more pronounced neuroendocrine response to a second bout of exercise on the same day compared with a first/single bout, involving both the sympatho-adrenal system and the hypothalamo-pituitary-adrenal axes.