The aim of this study was to analyze pathophysiological changes after testicular sperm aspiration (TESA) and microsurgical epididymal sperm aspiration (MESA) procedures. Twenty four mature male Wistar albino rats with a proven breeding history, weighing approximately 200-250 gm were used for the study. Animals were randomly divided into four groups (n = 6), i.e., control, sham-control, unilateral TESA, and MESA. Using a 22G needle, the aspiration procedures were done in testis or caudal epididymis. At the end of 60 days of survival, blood samples were collected and processed for antisperm antibody detection by enzyme-linked immunosorbent assay (ELISA). After euthanasia, testes and epididymides were collected and processed for paraffin embedding. Sections were stained with hematoxylin and eosin, and TUNEL technique. Serum antisperm antibody titer significantly increased in TESA (P < 0.001) when compared to MESA. Histomorphometric analysis indicated testicular alterations in TESA and MESA, with significant damage in TESA in both testes (P < 0.001). Following the MESA procedure, ipsilateral caudal and carpus epididymis showed significant alterations (P < 0.001) and no such alterations were seen in the ipsilateral caput and intact contralateral epididymis. TUNEL staining revealed an up-regulation of apoptosis in both contra- and ipsilateral testes of TESA. Needle prick had produced drastic and irreversible alterations in testis of TESA. Ensuing processes of immunological and inflammatory reaction had the potential to disrupt spermatogenesis and increase germ cell apoptosis. However, extrapolating conclusions from the experimental model to the clinic needs to be done cautiously.