Testicular sperm extraction (TESE) may provide spermatozoa for attempts at fertility with assisted reproduction; however, the physiological effects of TESE on testicular function are not well understood. In order to evaluate the effects of TESE on the testis, 64 patients were evaluated after TESE for non-obstructive azoospermia with physical examinations, serial scrotal sonography, histological analyses and evaluation of the success of repeated sperm retrieval attempts. At 3 months after TESE, 82% of evaluated patients had ultrasonographic abnormalities in the testis suggesting resolving inflammation or haematoma at the biopsy site. By 6 months, these acute inflammatory changes typically resolved leaving linear scars or calcifications. Two patients had documented impaired testicular blood flow, with complete devascularization of the testis for one patient after TESE with multiple biopsies. Repeat TESE procedures were far more likely to retrieve spermatozoa if the second TESE attempt was performed >6 months after the initial TESE procedure (80%), relative to those performed within 6 months (25%). Transient adverse physiological effects are common in the testis for up to 6 months after TESE. In addition, permanent devascularization of the testis can occur following TESE procedures with multiple biopsies. The risk of this complication may be minimized by using an open biopsy technique with optical magnification to directly identify testicular vessels.