Recently, the World Health Organization recommendation for abstinence time for semen analysis has been challenged in some studies and many of them have supported the advantages of a second short abstinence ejaculation. More evidence is needed to approve this for clinical use. This study aimed to compare the average routine abstinence time (2–7 days) with the short time (1–2 h) on sperm quality based on functional parameters in a population of oligo-astheno-teratozoospermia (OAT) men. The semen samples were retrieved from 50 men with OAT two times: one standard 2–7 days (long ejaculation) and short duration trimming (1–2 hours later the first ejaculation). All semen parameters as well as sperm DNA integrity were compared between groups. Results showed that mean sperm concentration (10.40 vs. 8.76), total sperm count (28.53 vs. 12.24) and mean semen volume (2.69 vs. 1.40) were higher in the first ejaculation (2–7 days of abstinence), while progressive motility (20.52 vs. 13.32), non-progressive motility (53.46 vs. 48.86), morphology (2.46 vs. 1.46) and viability (83.90 vs. 77.96) were significantly higher in the second ejaculation (P < 0.05). The second sample also showed lower immotile (26.82 vs. 38.02) and DNA fragmentation (19.5 vs. 26.96) (P < 0.05). Taking all data into account, an additional short abstinence period (AP) may be a simple and helpful strategy to obtain better sperm quality in couples with male infertility causes, especially in OAT patients. The recommended current guidelines regarding the AP may need to be revisited in severe male factors.