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This study aimed to determine whether limbs with a history of anterior cruciate ligament (ACL) injury reconstructed from the semitendinosus display different biceps femoris long head (BFlh) architecture and eccentric strength, assessed during the Nordic hamstring exercise, compared with the contralateral uninjured limb.The architectural characteristics of the BFlh were assessed at rest and at 25% of a maximal voluntary isometric contraction (MVIC) in the control group (n = 52) and in the group who had previous ACL injury (n = 15) using two-dimensional ultrasonography. Eccentric knee flexor strength was assessed during the Nordic hamstring exercise.Fascicle length was shorter (P = 0.001; d range, 0.90–1.31) and pennation angle (P range, 0.001–0.006; d range, 0.87–0.93) was greater in the BFlh of the ACL-injured limb compared with those in the contralateral uninjured limb at rest and during a 25% MVIC. Eccentric strength was lower in the ACL-injured limb when compared with the contralateral uninjured limb. Fascicle length, MVIC, and eccentric strength were not different between the left and right limb in the control group.Limbs with a history of ACL injury reconstructed from the semitendinosus have shorter fascicles and greater pennation angles in the BFlh compared with those of the contralateral uninjured side. Eccentric strength during the Nordic hamstring exercise of the ACL-injured limb is significantly lower than that of the contralateral side. These findings have implications for ACL rehabilitation and hamstring injury prevention practices, which should consider altered architectural characteristics.