Functional calibration methods were devised to improve repeatability and accuracy of the knee flexion-extension axis, which is used to define the medio-lateral axis of the femur coordinate system in gait analysis. Repeatability of functional calibration methods has been studied extensively in healthy individuals, but not accuracy in the absence of a benchmark knee axis. We captured bi-plane fluoroscopy data of the knee joint in 17 subjects with unilateral total knee arthroplasty during treadmill walking. The prosthesis provided a benchmark knee axis to evaluate the functional calibration methods. Stereo-photogrammetry data of thigh and shank marker clusters were captured simultaneously to investigate the effect of soft tissue artefact (STA). Three methods were tested, the Axis Transformation Technique (ATT) finds the best single fixed axis of rotation, 2DofKnee finds the axis that minimises knee varus-valgus and trajAJC finds the axis perpendicular to the trajectory, in the transverse plane of the femur, of a point located on the longitudinal axis of the tibia. Using fluoroscopy data, functional axes formed an angle of less than 2° in the transverse plane with the benchmark axis. True internal-external range of movement was correlated with decreased accuracy for ATT, while varus-valgus range of movement was correlated with decreased accuracy for 2DofKnee and trajAJC. STA had negative impact on accuracy and variability. Using stereo-photogrammetry data, the accuracy of 2DofKnee was 1.7°(SD: 5.1°), smaller than ATT 2.9°(SD: 5.1°) but not to trajAJC 1.7°(SD: 5.2°). Our results confirm that of previous studies, which utilised the femur condylar axis as reference.