Nowadays, a punch biopsy is a simple, reliable and inexpensive method for different types of tissue sampling. Equally, it is the method of choice for obtaining testicular tissue samples for pathohistological analysis and sperm for intracytoplasmic sperm injection. The results of this study clearly showed that a single biopsy has little effect on the biopsied testis, especially on total fertility. Triple biopsy showed by the same parameters that histological and immunohistochemical consequences were more significant compared to single but without a significant effect on overall fertility. Sperm analysis showed that single and triple biopsies did not have a significant effect on sperm count, motility and morphology. In addition, both single and triple punch biopsies of one testicle did not significantly affect the overall fertility potential of pubertal rats.