The functional roles of individual lower limb muscles during human walking may differ depending on walking speed or duration. In this study, 11 volunteers walked on a treadmill for 60 min at speeds corresponding to both optimal and 20% above optimal energetic cost of transport whilst oxygen consumption and medial gastrocnemius (MG) and soleus fascicle lengths were measured. Although energetic cost of transport was ∼12% higher at the faster speed, it remained constant over 60 min at both speeds, suggesting that humans can walk for prolonged periods at a range of speeds without compromising energetic efficiency. The fascicles of both muscles exhibited rather 'isometric' behaviour during the early to mid stance phase of walking, which appears to be independent of walking speed or movement efficiency. However, several functional differences were observed between muscles. MG exhibited time- and speed-dependent decreases in operating length, and shortened faster during the pushoff phase at the faster walking speed. Conversely, soleus exhibited consistent contractile behaviour regardless of walking speed or duration, and always shortened slower than MG during pushoff. Soleus appears to play a more important functional role than MG during walking. This may be especially true when walking for prolonged periods or at speeds above the most energetically efficient, where the force potential and thus the functional importance of MG appears to decline.