Various studies have sought to determine the typical v. optimal abstinence period after which semen samples should be collected, with many contradictory results reported. Several factors influence the semen microenvironment, and thus sperm parameters. In this study we focused on the secretions of the prostate, seminal vesicles and the epididymis. Semen samples were obtained from healthy normozoospermic males (n=16) after 4-day and 4-h periods of ejaculatory abstinence, and standard semen analysis was performed using computer-aided sperm analysis, whereas seminal plasma citric acid, neutral α-glucosidase and fructose concentrations were measured using assay kits. There were significant decreases in total sperm count (P<0.001), sperm concentration (P<0.05) and semen volume (P<0.05) after 4h compared with 4 days ejaculatory abstinence. Furthermore, increases were observed in total sperm motility (P<0.05) and sperm progressive motility (P<0.01) after a 4-h abstinence period, accompanied by significant reductions in citric acid (P<0.05), α-glucosidase (P<0.01) and fructose (P<0.01) concentrations. In addition, due to the decreased number of spermatozoa, these concentrations translated to a significant decrease in fructose (P<0.05) per spermatozoon, indicating an intrinsic mechanism capitalising on alternative sources of energy for increased metabolic function and subsequent sperm motility.