According to World Health Organization guidelines, ejaculatory abstinence (EA) of 2 to 7 days is recommended for semen analysis. This study aimed to determine how seminal quality may be affected by two EA periods from the same man. We evaluated seminal samples from 65 men by conventional semen analysis and qualitative characteristics after 1 day and 4 days of EA (two samples/man). We qualitatively analyzed the semen by examining oxidative activity (intracellular and seminal plasma), sperm function (acrosome integrity, mitochondrial activity, and nuclear DNA integrity), and epididymal function. As expected, samples collected after 1 day of EA showed a decrease in volume and sperm total number compared to samples collected after 4 days of EA. The sperm motility of the samples collected after 1 day of EA was better compared to samples collected after 4 days of EA. Oxidative activity measured was lower after 1 day of EA compared to those measured after 4 days of EA. Regarding sperm function, samples collected after 1 day of EA showed an increase in acrosome integrity, mitochondrial activity, and nuclear DNA integrity compared to samples collected after 4 days of EA. Epididymal function showed no difference between the two-time points. Although samples collected after 4 days of EA showed better results concerning sperm quantity, samples collected after 1 day of EA showed better qualitative results, including motility, oxidative activity, and sperm function. Thus, we conclude that sperm storage at the epididymal tail may make spermatozoa more susceptible to oxidative damage.