According to the prescribed guidelines of the World Health Organization, subjects must remain abstinent for a minimum period of 48 hours, but not longer than 7 days before collecting a semen sample for standard analysis. [1] However, the basis for this recommendation remains uncertain. Various studies have investigated the typical time after which human semen samples should be collected for standardized analysis and have shown contradictory results [2, 3, 4]. This study aimed to investigate the effect of short and long term abstinence on sperm kinematics in young healthy individuals. Semen samples (n=100) were collected from potentially fertile, healthy males (20 to 30 years). Donors abstained for 4 days prior to the first sample collection, while the second sample was collected from the same donor after 4 hours of abstinence. Sperm motility parameters were quantified by the Sperm Class Analyser (SCA) (Microptic, S.L., Barcelona, Spain). Motility and Kinematics parameters included total motility, progressive motility, curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), and amplitude of lateral head displacement (ALH). Semen pH was assessed with pH indicator paper (MERCK) with graduated colours indicating pH from 6.4 to 8.0. Sperm superoxide (O2-) levels were assessed by flow cytometry, using dihydroethidium (DHE). Data were analysed using Paired Student’s t-tests on Graph Pad Prism™ software and presented as Mean ± S.E.M, significance was set at p < 0.05. A significant increase in pH (7.69±0.01 vs. 7.58±0.016, p = 0.0001), total motility (62.29±1.53 vs. 58.86±1.51 p = 0.0027), progressive motility (49.58%±1.47 vs. 44.98%±1.37, p = 0.0001), VCL (81.99µm/s± 1.68 vs. 76.24µm/s±1.08, p = 0.0001), VSL (32.90µm/s±0.61 vs. 29.81µm/s± 0.50, p = 0.0001) and LIN (40.89%±0.79 vs. 39.42%±0.59, p = 0.0110) was observed in the second sample compared to the first sample. No significant differences were observed in ALH and O2-. The beneficial effect of a short abstinence period (4hr) on sperm movement characteristics was accompanied by increased pH, but appeared to be independent of O2- levels. The clinical significance of this finding would be that short abstinence prior to sample collection may improve the outcomes of ART and fertility preservation.