We have demonstrated that sexual activity produces transient sympathoadrenal activation and a pronounced, long-lasting increase in prolactin in men and women. However, by analyzing endocrine alterations at 10-min intervals, a precise assignment of these changes to the pre-, peri- and postorgasmic periods was not possible. Thus, the current study aimed to accurately differentiate the endocrine response to sexual arousal and orgasm in men using an automatic blood collection technique with 2-min sampling intervals. Blood was drawn continuously before, during and after orgasm over a total period of 40 min in 10 healthy subjects and were compared with samples obtained under a control condition. Sexual activity induced transient increases of plasma epinephrine and norepinephrine levels during orgasm with a rapid decline thereafter. In contrast, prolactin levels increased immediately after orgasm and remained elevated throughout the experiment. Although oxytocin was acutely increased after orgasm, these changes were not consistent and did not reach statistical significance. Vasopressin, LH, FSH and testosterone plasma concentrations remained unaltered during sexual arousal and orgasm. These data confirm that prolactin is secreted after orgasm and, compared with oxytocin, seems to represent a more reliable and sustained marker for orgasm in man. The results further reinforce a role for prolactin either as a neuroendocrine reproductive reflex or as a feedback mechanism modulating dopaminergic systems in the central nervous system that are responsible for appetitive behavior.