Background Few studies compared the length change of ligaments of normal knees during dynamic activities of daily living. The aim of this study was to investigate in vivo length change of ligaments of the normal knees during high flexion. Methods Eight normal knees were investigated. Each volunteer performed squatting, kneeling, and cross-leg motions. Each sequential motion was performed under fluoroscopic surveillance in the sagittal plane. The femoral, tibial, and fibular attachment areas of the anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), deep medial collateral ligament (dMCL), superficial medial collateral ligament (sMCL), and lateral collateral ligament (LCL) were determined according to osseous landmarks. After 2D/3D registration, the direct distance from the femoral attachment to the tibial or fibular attachment was measured as the ligament length. Results From 20° to 90° with flexion, the ACL was significantly shorter during cross-leg motion than during squatting. For the PCL, dMCL, sMCL, and LCL, there were no significant differences among the 3 motions. Conclusion The ACL was shorter during cross-leg motion than during squatting in mid-flexion. This suggests that the ACL is looser during cross-leg motion than during squatting. On the other hand, the length change of the PCL, MCL, and LCL did not change even though the high flexion motions were different.