Measurement of the changes of the three-dimensional (3D) motion and surface kinematics of the knee under different external loading conditions is essential for the understanding and evaluation of the function of the joint, as well as for relevant clinical applications. Knee extension exercise (KEE) has been applied extensively in the rehabilitation programs of patients with various knee disorders. This study measured the 3D knee kinematics of eight normal subjects during active knee extension for unloaded and loaded conditions using a voxel-based method for the registration of fluoroscopic images with CT bone data. The knee kinematics during unloaded conditions were found to be similar to previous findings. A mass of 5 kg at the ankle did not affect the joint angles but significantly altered the lateral contact positions during knee extension, especially at knee flexion angles higher than 75 degrees, and also reduced the asymmetry of the surface kinematics between the medial and lateral condyles. The results of the current study may be useful for knee replacement design and for developing guidelines for the use of KEEs for the rehabilitation of patients with knee disorders.