Objective studies of men's reproductive function are hindered by their reliance on: (i) self-reporting to quantify sexual activity and (ii) masturbation to quantify sperm output rendering both types of estimate vulnerable to unverifiable subjective factors. We therefore examined whether detection of spermatozoa and measurement of prostate-specific antigen (PSA) in urine could provide objective semiquantitative estimates of sperm output and recent ejaculation, respectively, using widely available laboratory techniques. Of 11 healthy volunteers who provided urine samples before and at intervals for 5 days after ejaculation, sperm was present in 2/11 men before, and in all 11/11 samples immediately after ejaculation, but by the second and subsequent void, spermatozoa were present in ,10%. PSA was detectable at high levels in all urine samples, peaking at the first post-ejaculatory sample but returning to baseline levels by the second post-ejaculatory void. We conclude that urinary spermatozoa and PSA are objective biomarkers for sperm production and sexual activity, but only for a short-time window until the first post-ejaculatory urine void. Hence, for a single urine specimen, the presence of spermatozoa and PSA are valid biomarkers, reflecting sperm production and recent ejaculation only until the next micturition, so their measurement should be restricted to the first morning urine void. Keywords: ejaculation; PSA; sexual activity; sperm output; spermatozoa; urine INTRODUCTIONMajor limitations for objective studies of human male reproductive function are the reliance on self-reporting of sexual activity and on masturbation to provide semen samples for analysis of sperm output. Both intrude on sensitive personal matters and rely wholly or partly on subjective aspects of sexual activity. Self-reporting of sexual activity is influenced by complex personal attitudes and perceptions and is effectively impossible to corroborate. Hence, such data are subject to unverifiable bias or distortion. Similarly, the only objective measure of sperm output requires semen specimen collected by masturbation, usually a sensitive and private activity. As a result, men are reluctant to participate in studies requiring semen samples unless actively concerned about their fertility, creating a major participation bias.