A muscle’s physiological properties determine its force-length (F-L) and force-velocity (F-V) relationships, and thus the fibre length and contraction speed at which maximal force and power can be produced. Here, we investigated the relationship between F-L and F-V relationships and in vivo muscle function in the guinea fowl (Numida Meleagris) main ankle extensor, the lateral gastrocnemius (LG) muscle during walking and running. To do so, we map each individual birds’ in vivo work loops to their individual force-length and force-velocity relationships. We hypothesize that muscles operate near optimal conditions for force and power production, respectively, during walking and running tasks to optimize efficiency during locomotion. We found that force development occurs entirely on the ascending limb of the F-L relationships curve in both walking and running and thus not at lengths that would maximize force output. LG muscles operate at low velocities at peak force and at optimal intermediate velocities during lower forces. These in vivo LG velocities are consistent with optimizing force and power according to the F-V relationship. Hence, a muscle’s operating length and velocity are not solely driven by efficiency. A potential explanation is that muscles act on the ascending limb of the F-L relationship to improve robustness against perturbations. We are currently performing model-based simulations of guinea fowl locomotion to explore how a muscle’s physiological properties affect its operating length and velocity. Our central hypothesis is that we will need to account for robustness against uncertainty in the simulations to capture the experimentally observed relationship.