In this study, we investigated differences in electromyographic activity in the biceps femoris long head (BFl), semitendinosus, and semimembranosus muscles during 45° hip extension with different knee angles during eccentric, concentric, and isometric hip 0°, and isometric hip 45° conditions with non-external resistance and 5-kg load. Twenty-two male volunteers performed 45° hip extension with knee flexion angles of 0°, 45° and 90° with non-external resistance and 5-kg load eccentric, concentric, isometric hip 0°, and isometric hip 45° conditions. The electromyographic data obtained during each condition were normalized with the values collected during maximal voluntary isometric contraction of each muscle. A multivariate analysis of variance with repeated measures using syntax was used to compare the normalized electromyography of each muscle across different knee joint angles in each weight condition. Electromyographic activities of the BFl, semitendinosus, and semimembranosus at 45° and 90° knee flexion angles were significantly greater than at 0° in the eccentric, concentric, isometric hip 0°, and in isometric hip 45° conditions with both non-external resistance and 5-kg load (p<0.05), except for that of the BFl and semitendinosus in isometric hip 45° with 5-kg load. The electromyographic activity of the BFl was significantly higher than that of the semimembranosus at 90° knee flexion in all conditions (p < 0.05), except during eccentric with non-external resistance. There was no significant difference in electromyographic activity in the hamstring muscles among different knee angles and muscular contraction conditions. This study showed that 45° hip extension with 45° and 90° knee flexion might be better in terms of the recruiting hamstring activity compared to 0° knee flexion, regardless of the training intensity. We recommend 45° hip extension exercises with knee flexion angles of 45° and 90° to activate the BFl, in preventing hamstring strain.