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Total knee arthroplasty (TKA) is a procedure with function dependent upon correct tensioning of the soft-tissue constraints. The purpose of this study was to examine the length-change behaviour of the collateral ligaments during knee flexion–extension before and after TKA. The influence of differing degrees of internal-external rotation of the femoral component on slackening/tightening of the collateral ligaments during knee flexion was to be studied. The length-change patterns of the collateral ligaments were measured in eight intact knees in vitro: sutures were passed along the ligaments and attached to displacement transducers. Measurements were repeated after TKA with the femoral component in neutral rotation, then with 5° internal and 5° external rotation. Both the MCL and LCL slackened during knee flexion from 0° to 110° flexion, at all stages of the experiment. In the native knee, the MCL slackened 2 mm, whilst the LCL slackened 7 mm. The MCL slackened a further 3 mm and the LCL a further 4 mm during flexion post-TKA. A 5° external rotation of the femoral component slackened the MCL 2 mm more and tightened the LCL by 2 mm. The opposite effects resulted from 5° internal rotation. The collateral ligaments slackened more than normal following TKA, and these length changes were increased by femoral component rotation. External rotation of the femoral component to address patellar tracking may slacken the MCL and thus lead to valgus instability in the flexed knee.