The mechanical advantage of the knee extensor mechanism depends heavily on the patellar tendon moment arm (PTMA). Understanding which factors contribute to its variation may help improve functional outcomes following arthroplasty. This study optimized PTMA measurement, allowing us to quantify the contribution of different variables. The PTMA was calculated about the instantaneous helical axis of tibiofemoral rotation from optical tracked kinematics. A fabricated knee model facilitated calculation optimization, comparing four data smoothing techniques (raw, Butterworth filtering, generalized cross-validated cubic spline-interpolation and combined filtering/interpolation). The PTMA was then measured for 24 fresh-frozen cadaveric knees, under physiologically based loading and extension rates. Combined filtering/interpolation enabled sub-mm PTMA calculation accuracy throughout the range of motion (root-mean-squared error 0.2 mm, max error 0.4 mm), whereas large errors were measured for raw, filtered-only and interpolated-only techniques at terminal flexion/extension. Before scaling, the mean PTMA was 46 mm; PTMA magnitude was consistently larger in males (mean differences: 5 to 10 mm, p < .05) and was strongly related to knee size: larger knees have a larger PTMA. However, while scaling eliminated sex differences in PTMA magnitude, the peak PTMA occurred closer to terminal extension in females (female 15°, male 29°, p = .01). Knee size accounted for two-thirds of the variation in PTMA magnitude, but not the flexion angle where peak PTMA occurred. This substantial variation in angle of peak PTMA has implications for the design of musculoskeletal models and morphotype-specific arthroplasty. The developed calculation framework is applicable both in vivo and vitro for accurate PTMA measurement.