OBJECTIVE: This systematic review and meta-analysis was conducted to clarify the effects of abstinence duration on semen quality in human studies. METHODS: This review was prospectively registered in PROSPERO and conducted in accordance with PRISMA 2020 guidelines. PubMed (MEDLINE), Scopus, and Web of Science were searched from inception to the most recent update. Human studies evaluating ejaculatory abstinence duration in relation to semen parameters were included. For methodological clarity, studies were classified into (1) single-ejaculate abstinence-comparison studies (paired or independent-group designs) and (2) repeated or frequent ejaculation studies involving consecutive ejaculations. Only single-ejaculate abstinence-comparison studies contributed to the primary meta-analyses. Random-effects meta-analyses were performed for semen volume, sperm concentration, total sperm count, total sperm motility, progressive sperm motility, and sperm morphology. Subgroup and sensitivity analyses were conducted, and risk of bias was assessed using ROBINS-I. RESULTS: Eighty-two studies were included in the qualitative synthesis, of which 59 contributed to the quantitative meta-analyses. Comparisons involving longer abstinence intervals (e.g., ≥ 4-5 days compared with 2-3 days) were associated with significantly higher semen volume (MD - 1.07 mL, indicating higher volume with longer abstinence) and total sperm count (MD - 80.61 × 10⁶ sperm per ejaculate). In contrast, very short abstinence intervals were associated with significantly higher total sperm motility (MD 4.37%) and progressive sperm motility (MD 3.24%). No consistent overall differences were observed for sperm concentration or morphology across abstinence-interval contrasts. Substantial heterogeneity was observed across outcomes; however, sensitivity analyses generally produced similar directions of association, although substantial heterogeneity remained. The certainty of evidence was low or very low across outcomes, mainly because of substantial heterogeneity, residual confounding, and imprecision. CONCLUSIONS: Across explicit abstinence-interval contrasts, higher-duration intervals were associated with greater semen volume and total sperm count, whereas lower-duration intervals were associated with higher total and progressive sperm motility. The certainty of evidence was low or very low, and these results represent laboratory associations rather than causal effects or universally applicable clinical thresholds. Repeated or frequent ejaculation protocols and reproductive outcomes were synthesized narratively. Abstinence recommendations should therefore be individualized according to the purpose of semen collection and the clinical context.