Human feet have evolved to facilitate bipedal locomotion, losing an opposable digit that grasped branches in favor of a longitudinal arch (LA) that stiffens the foot and aids bipedal gait. Passive elastic structures are credited with supporting the LA, but recent evidence suggests that plantar intrinsic muscles (PIMs) within the foot actively contribute to foot stiffness. To test the functional significance of the PIMs, we compared foot and lower limb mechanics with and without a tibial nerve block that prevented contraction of these muscles. Comparisons were made during controlled limb loading, walking, and running in healthy humans. An inability to activate the PIMs caused slightly greater compression of the LA when controlled loads were applied to the lower limb by a linear actuator. However, when greater loads were experienced during ground contact in walking and running, the stiffness of the LA was not altered by the block, indicating that the PIMs’ contribution to LA stiffness is minimal, probably because of their small size. With the PIMs blocked, the distal joints of the foot could not be stiffened sufficiently to provide normal push-off against the ground during late stance. This led to an increase in stride rate and compensatory power generated by the hip musculature, but no increase in the metabolic cost of transport. The results reveal that the PIMs have a minimal effect on the stiffness of the LA when absorbing high loads, but help stiffen the distal foot to aid push-off against the ground when walking or running bipedally.