Contradictions exist between studies of the 3D kinematics of the knee. We hypothesize that they are in part due to differences in the gesture performed by the subjects during kinematic assessment. The purpose of this study is to evaluate the impact of gesture variations on knee kinematics. Seventeen healthy male subjects performed 20-s series of knee-bends in a knee-bend standardizing structure. All series differed regarding either foot rotation, knee excursion, or hip rotation. 3D knee kinematics were recorded using optical position sensors mounted on a skin-motion-reducing harness. Kinematic comparisons were made between a gesture of reference (the standard gesture) and every other gesture. Analyses were performed on average differences. Differences of up to 15° of tibial rotation were found for gestures involving different foot rotation. Gestures involving different knee excursion brought on differences of more than 4° of tibial rotation while hip rotation induced more than 5° of tibial rotation. It is hereby demonstrated that gesture differences can have a dramatic impact on measured knee kinematics. Hence gesture performance needs to be carefully monitored during 3D kinematic assessment of the weight-bearing human knee.