A central question in biology is how animals successfully behave under complex natural conditions. Although changes in locomotor behaviour, motor control and force production in relation to incline are commonly examined, a wide range of other factors, including a range of perch diameters, pervades arboreal habitats. Moving on different substrate diameters requires considerable alteration of body and limb posture, probably causing significant shifts in the lengths of the muscle–tendon units powering locomotion. Thus, how substrate shape impacts in vivo muscle function remains an important but neglected question in ecophysiology. Here, we used high-speed videography, electromyography, in situ contractile experiments and morphology to examine gastrocnemius muscle function during arboreal locomotion in the Cuban knight anole, Anolis equestris. The gastrocnemius contributes more to the propulsive effort on broad surfaces than on narrow surfaces. Surprisingly, substrate inclination affected the relationship between the maximum potential force and fibre recruitment; the trade-off that was present between these variables on horizontal surfaces became a positive relationship on inclined surfaces. Finally, the biarticular nature of the gastrocnemius allows it to generate force isometrically, regardless of substrate diameter and incline, despite the fact that the tendons are incapable of stretching during cyclical locomotion. Our results emphasize the importance of considering ecology and muscle function together, and the necessity of examining both mechanical and physiological properties of muscles to understand how animals move in their environment.