Background & Objective: The effect of storage time and temperature on the prepared semen sample was evaluated, but the optimal condition is unclear. The aim of this study was to assess the effect of long-term incubation of prepared sperm at testicular temperature versus room temperature on semen parameters and DNA fragmentation index (DFI).Materials & Methods: Sperm samples were collected from 40 patients between 2019 and 2020. Each sample was separated into two parts and underwent a non-direct swimup method. One group was placed in a 35°C incubator, and the other group was kept at room temperature (26°C) in the dark. Both groups were evaluated at intervals of 45 minutes, 24 hours and 48 hours after sampling in terms of sperm concentration, motility, morphology, and DFI. Student t-test and repeated measures analysis of variance were used.Results: Sperm count (P=0.007) and motility (P<0.001) at 26°C in three-time intervals of 45 minutes, 24 hours and 48 hours were significantly higher than 35°C. The proportion of normal morphology spermatozoa at 26 and 35°C at 45 min, 24 h, and 48 h did not show a significant difference (P=0.08). DFI at 26°C in three-time intervals was significantly lower than 35°C (P=0.008). Conclusion:The results of this study indicated that when the prepared sperm samples are incubated for 24 h at 26°C compared to 35°C, they show significantly better quality and good quality of sperm can be retained for several hours if stored at room temperature.