On the basis of differences in physiology, e.g., histochemical properties and spindle density, and the structural design of the cat soleus (SO) and medial gastrocnemius (MG) muscles, we hypothesized that 1) fascicle length changes during overground walking would be both muscle and slope dependent, which would have implications for the muscles' force output as well as sensory function, and that 2) muscle-tendon unit (MTU) and fascicle length changes would be different, in which case MTU length could not be used as an indicator of muscle spindle strain. To test these hypotheses, we quantified muscle fascicle length changes and compared them with length changes of the whole MTU in the SO and MG during overground walking at various slopes (0, ± 25, ± 50, +75, and +100%). The SO and MG were surgically instrumented with sonomicrometry crystals and fine-wire electromyogram electrodes to measure changes in muscle fascicle length and muscle activity, respectively. MTU lengths were calculated using recorded ankle and knee joint angles and a geometric model of the hindlimb. The resultant joint moments were calculated using inverse dynamics analysis to infer muscle loading. It was found that although MTU length and velocity profiles of the SO and MG appeared similar, length changes and velocities of muscle fascicles were substantially different between the two muscles. Fascicle length changes of both SO and MG were significantly affected by slope intensity acting eccentrically in downslope walking (-25 to -50%) and concentrically in upslope walking (+25 to +100%). The differences in MTU and fascicle behaviors in both the SO and MG muscles during slope walking were explained by the three distinct features of these muscles: 1) the number of joints spanned, 2) the pennation angle, and 3) the in-series elastic component. It was further suggested that the potential role of length feedback from muscle spindles is both task and muscle dependent. Copyright © 2009 the American Physiological Society.