BackgroundLimited studies have compared the muscle activity of the medial and lateral hamstrings as knee flexors with tibial internal and external rotation and hip extensors with hip internal and external rotation. In particular, hamstring activity during hip extension with hip rotation has rarely been investigated. ObjectiveThis study aimed to compare the muscle activity of the medial and lateral hamstrings as knee flexors and hip extensors and to compare the activity of these muscles according to tibial rotation during isometric knee flexion and hip rotation during isometric hip extension. MethodsA total of 23 healthy adults participated in the study. The electromyographic (EMG) activity of the hamstrings was measured during maximal isometric knee flexion and maximal isometric hip extension. In addition, tibial rotation was applied actively during maximal isometric knee flexion, whereas hip rotation was applied actively during maximal isometric hip extension. ResultsEMG activity during maximal isometric knee flexion with tibial internal and external rotation was significantly higher than that during maximal isometric hip extension with hip internal and external rotation, respectively. For EMG activity according to tibial and hip rotation, there was no significant difference between tibial internal and external rotation during maximal isometric knee flexion, whereas there was a significant difference between hip internal and external rotation during maximal isometric hip extension. ConclusionHamstring activity was higher for knee flexors than for hip extensors. However, hip rotation during maximal isometric hip extension is an effective intervention for selective muscle activation of the medial and lateral hamstrings.