Objective To build a method to calculate hamstring muscle optimal length in vivo, and compare with other indirect parameters which represent the optimal length. Methods The knee ?exion torque and kinematic data were synchronously recorded. Musculoskeletal model of lower limbs was built to obtain hamstring strength and muscle length, and then hamstring muscle optimal lengths were calculated. Results Knee flexion angle at peak knee flexion torque was signi?cantly greater than flexion angle at peak hamstring strength and the correlation coefficient between them was 0.741. Hamstring muscle optimal lengths were signi?cantly greater than corresponding mus-cle lengths in standing and the correlation coefficient between them were low. Conlusions The calculation method of hamstring muscle optimal length in this study sets a basis for future studies on injury mechanism and risk factors. Knee flexion angle at peak knee flexion torque could partly represent hamstring optimal length. Hamstring muscle length in standing should not be used as an approximation of hamstring optimal length.