PURPOSE To measure the in vivo anterolateral ligament (ALL) length change in healthy knees during step-up and sit-to-stand motions. METHODS Eighteen healthy knees were imaged using magnetic resonance and dual fluoroscopic imaging techniques during a step-up and sit-to-stand motion. The ALL length change was measured using the shortest three-dimensional wrapping path, with its femoral attachment located slightly anterior-distal (ALL-Claes) or posterior-proximal (ALL-Kennedy) to the fibular collateral ligament attachment. The ALL length measured from the extended knee position of the non-weight-bearing magnetic resonance scan was used as a reference to normalize the length change. RESULTS During the step-up motion (approximately 55° flexion to full extension), both the ALL-Claes and ALL-Kennedy showed a significant decrease in length of 21.2% (95% confidence interval 18.0-24.4, P < .001) and 24.3% (20.6-28.1, P < .001), respectively. During the sit-to-stand motion (approximately 90° flexion to full extension), both the ALL-Claes and ALL-Kennedy showed a consistent, significant decrease in length of 35.2% (28.8-42.2, P < .001) and 39.2% (32.4-46.0, P < .001), respectively. From approximately 90° to 70° of flexion, a decrease in length of approximately 6% was seen; 70° of flexion to full extension resulted in an approximately 30% decrease in length. CONCLUSIONS The ALL was found to be a nonisometric structure during the step-up and sit-to-stand motion. The length of the ALL was approximately 35% longer at approximately 90° of knee flexion when compared with full extension and showed decreasing length at lower flexion angles. Similar ALL length change patterns were found with its femoral attachment located slightly anterior-distal or posterior-proximal to the fibular collateral ligament attachment. CLINICAL RELEVANCE These data suggest that, if performing anatomic ALL reconstruction, graft fixation may be performed beyond 70° flexion to reduce the chance of lateral compartment overconstraint. Anatomic ALL reconstruction may affect the knee kinematics more in high flexion than at low flexion angles.