The region over which skeletal muscles operate on their force-length (F-L) relationship is fundamental to the mechanics, control and economy of movement. Yet surprisingly little experimental data exist on normalized length operating ranges of muscle during human gait, or how they are modulated when mechanical demands (such as force output) change. Here we explored the soleus muscle (SOL) operating lengths experimentally in a group of healthy young adults by combining subject-specific F-L relationships with in vivo muscle imaging during gait. We tested whether modulation of operating lengths occurred between walking and running, two gaits that require different levels of force production and different muscle-tendon mechanics, and examined the relationship between optimal fascicle lengths (L(0)) and normalized operating lengths during these gaits. We found that the mean active muscle lengths reside predominantly on the ascending limbs of the F-L relationship in both gaits (walk, 0.70-0.94 L(0); run, 0.65-0.99 L(0)). Furthermore, the mean normalized muscle length at the time of the peak activation of the muscle was the same between the two gaits (0.88 L(0)). The active operating lengths were conserved, despite a fundamentally different fascicle strain pattern between walking (stretch-shorten cycle) and running (near continuous shortening). Taken together, these findings indicate that the SOL operating length is highly conserved, despite gait-dependent differences in muscle-tendon dynamics, and appear to be preferentially selected for stable force production compared with optimal force output (although length-dependent force capacity is high when maximal forces are expected to occur). Individuals with shorter L(0) undergo smaller absolute muscle excursions (P<0.05) so that the normalized length changes during walking and running remain independent of L(0). The correlation between L(0) and absolute length change was not explained on the basis of muscle moment arms or joint excursion, suggesting that regulation of muscle strain may occur via tendon stretch.