Limb asymmetries, as determined through in vivo biomechanical measures, are known risk factors for anterior cruciate ligament (ACL) injury. Previous cadaveric studies have shown a lack of significant differences in ligament strain between contralateral lower extremities when identical kinematics were simulated on specimens. Recent methodological developments have applied in vivo knee kinetics to exert landing forces on cadaveric lower extremities to mimic ACL injury events, but it is unknown whether contralateral limbs fail in a consistent manner during impact simulator testing.It was hypothesized that contralateral lower extremities would not exhibit side-to-side differences in ligament strains. Furthermore, it was hypothesized that failure loads and failure locations would be independent of limb dominance.Controlled laboratory study.Fourteen pairs of cadaveric lower extremities were obtained from an anatomic donations program (8 female, 6 male; mean ± SD: age, 41.7 ± 8.1 years; mass, 86.8 ± 27.0 kg; body mass index, 29.4 ± 9.0 kg/m). A mechanical impact simulator was used to re-create the impulse ground-reaction force generated during an in vivo landing task. Ligament strains were recorded by differential variable force transducers implanted on the ACL and medial collateral ligament (MCL).No significant differences were observed in peak ACL or peak MCL strain for 5 loading conditions. Fisher exact tests of independence revealed that limb dominance was independent of both load at failure and failure location.There were no significant differences in ACL and MCL strain values between limb sides during in vitro impact simulation testing. This finding indicates that limb dominance does not influence the failure threshold of the ACL, since there was no significant difference in failure strains. The functional mechanics of the ACL are comparable between contralateral pairs from the same healthy specimen.Injury mechanisms and intra-articular mechanics cannot be ethically studied in an in vivo setting. The current study provides additional insight into limb asymmetry that is observed among athletes in clinical sports medicine settings.