Introduction: The changes in knee axial rotation play an important role in traumatic and non-traumatic knee disorders. It is known that support afferentation can affect the axial rotator muscles. The condition of innervation of the semitendinosus (ST) and biceps femoris posterior (BFp) has changed in non-terrestrial and terrestrial vertebrates in evolution; thus, we hypothesized this situation might be replayed by hindlimb unloading (HU). Methods: In the present study, the EMG activity of two hamstring muscles, m. ST and m. BFp, which are antagonists in axial rotation of the tibia, was examined before and after 7 days of HU. Results: During locomotion and swimming, the ST flexor burst activity increased in the stance-to-swing transition and in the retraction-protraction transition, respectively, while that of BFp remained unchanged. Both ST and BFp nonburst extensor activity increased during stepping and decreased during swimming. Conclusions: Our results show that (1) the flexor burst activity of ST and BFp depends differently on the load-dependent sensory input in the step cycle; (2) shift of the activity gradient towards ST in the stance-to-swing transition could produce excessive internal tibia torque, which can be used as an experimental model of non-traumatic musculoskeletal disorders; and (3) the mechanisms of activity of ST and BFp may be based on reciprocal activity of homologous muscles in primary tetrapodomorph and depend on the increased role of supraspinal control.