This was an experimental study to determine the influence of short abstinence time on sperm motility, morphology and DNA damage performed at the Assisted Reproductive Technology (ART) unit, Ramathibodi Hospital. Fifty-seven semen samples with normal semen analysis according to World Health Organization (WHO) 1999 and Kruger strict criteria were obtained. Volunteers were instructed to abstain for 3-5 days, 18-30 hours and 4 days for the 1st, 2nd and 3rd semen collection Total motile sperm count (TMSC) was determined using computer-assisted semen analysis (CASA); sperm morphology was determined using eosin-methylene blue staining and DNA damage was assessed by TUNEL assay. The results showed that TMSC was not significantly different between the 1st (3-5 days abstinence time) and 2nd semen collection (18-30 hours abstinence time after the 1st collection) (p value=0.289). The percentage of DNA damage (4.6% vs. 9.8%, p value<0.001) and normal morphological sperm (14.9% vs. 17.2%, p value<0.001) in the 2nd semen collection were significantly lower than the 1st collection, although the value was still in the normal range. Semen parameters of the 3rd collection (4 days abstinence time after the 2nd collection) correlate with those of the 1st collection. We conclude that in normal semen, with an abstinence time of only one day, TMSC was not significantly different between 3-5 days abstinence time and 18-30 hours abstinence time. The result of DNA damage and the percentage of normal morphological sperm were significantly lower in 18-30 hours abstinence time.