Usage of open-kinetic-chain (OKC) or closed-kinetic-chain (CKC) exercises during rehabilitation planning after anterior cruciate ligament (ACL) reconstruction has been debated for decades. However, the ACL elongation pattern during different rehabilitation exercises at different loadings remains unclear. This study aimed to determine the effects of OKC and CKC exercises on the length of ACL anteromedial bundle (AMB) and posterolateral bundle (PLB) to provide biomechanical support for making rehabilitation schedules. Laboratory Descriptive Study. Eighteen healthy volunteers were asked to perform two OKC motions, including non-weight-bearing and 10 kg loaded seated knee extension (OKC-0, OKC-10), as well as two CKC motions, including box squat (BS) and deep single-legged lunge (Lunge). Techniques of 2D-to-3D image registration and 3D ligament simulation were used to quantify length changes of ACL. The motion which led to the least and most ACL elongation were OKC-0 and OKC-10, respectively. The AMB and PLB were significantly longer in OKC-10 than those in OKC-0 during 0-60° and 0-55° of knee flexion (p < 0.01). Compared with reference length, the AMB and PLB were stretched during 0-30° and 0-10° respectively during OKC-10. During CKC exercises, the AMB and PLB were also stretched from 0 to 25°and 0-5°, respectively. Additionally, no significant difference was found in the length change of ACL bundles between BS and lunge. OKC-0 may be safe for the rehabilitation program after ACL reconstruction, and loaded exercises shall be applied when restricted with >30° in early-stage rehabilitation.