Triceps surae muscles are mechanically connected by the shared Achilles tendon and by epimuscular myofascial connections. We aimed to assess the effects of proximal lengthening of gastrocnemius and plantaris muscles, imposed by changes in knee angle, on the magnitude and direction of the 3D ankle moment exerted by the soleus muscle and the mechanical interaction between ankle plantar flexor muscles during co-activation of gastrocnemius muscle, in the rat (N=9). Ankle angle was kept constant (90 deg), whereas knee angle was varied between 60 deg and 130 deg. At each knee angle, the soleus muscle was excited individually as well as simultaneously with the gastrocnemius muscle (SO+GA). The mathematical sum of individual soleus and gastrocnemius ankle moments was compared with the ankle moment exerted by SO+GA to assess nonlinear summation. Knee angle did not affect the magnitude of the soleus ankle moment (P=0.695). Moment directions in the transverse (P=0.050) and frontal (P=0.008) planes were affected by knee angle, but dissection indicated that this was not caused by length changes of the two-joint synergistic muscles. Nonlinear summation was found in the magnitude (-1.4±1.9%, mean±s.d., P<0.001) and in the frontal plane vector direction of the ankle moment (0.13±0.23 deg, P=0.003); however, the extent did not change with knee angle. Although contraction of SO+GA increased the length of the Achilles tendon compared with its length at rest, this was not dependent on the knee angle (P=0.649). Despite the fact that intermuscular force transmission per se cannot be excluded, we conclude that, in vivo, the mechanical effects of epimuscular myofascial connections between rat ankle plantar flexors are not functionally relevant.