Introduction. This study evaluates histological alterations in the hamstring tendon caused by cleaning the muscle fibers during preparation of an autograft for anterior cruciate ligament reconstruction. Methods. We collected semitendinosus tendons from 6 patients who underwent anterior cruciate ligament reconstruction and used a curette to scratch the tendon for cleaning. Three groups of increasing repetitions of scratching (5, 10, and 15) were compared to a control (no scratching). Histological alterations in the tendon were evaluated using the modified Bonar score. Immunohistochemical analysis was also performed for type I collagen, with measurement of the area stained for type I collagen and cell numbers in the collagen fibers. Results. The mean modified Bonar score was 0.8 in the control group, and 3.2, 5.7, and 7.2 for the 5-, 10-, and 15-repeated scratching groups, respectively. The area of type I collagen staining decreased with increasing number of scratches (P<0.05 for all groups), with a decrease of >50% in cellular density between the 5-and 10-repetition groups (P=0.004). Conclusion. Cleaning the hamstring tendons by scratching caused histological alterations and damage to type I collagen. Specifically, cellular density decreased markedly on scratching 10 times, suggesting that excessive curettage, leading to >10 scratches, might be deleterious.