Neuromuscular control loops feature substantial communication delays, but mammals run robustly even in the most adverse conditions.In vivoexperiments and computer simulation results suggest that muscles’ preflex—an immediate mechanical response to a perturbation—could be the critical contributor. Muscle preflexes act within a few milliseconds, an order of magnitude faster than neural reflexes. Their short-lasting action makes mechanical preflexes hard to quantifyin vivo. Muscle models, on the other hand, require further improvement of their prediction accuracy during the non-standard conditions of perturbed locomotion. Our study aims to quantify the mechanical work done by muscles during the preflex phase (preflex work) and test their mechanical force modulation. We performedin vitroexperiments with biological muscle fibers under physiological boundary conditions, which we determined in computer simulations of perturbed hopping. Our findings show that muscles initially resist impacts with a stereotypical stiffness response—identified as short-range stiffness—regardless of the exact perturbation condition. We then observe a velocity adaptation to the force related to the amount of perturbation similar to a damping response. The main contributor to the preflex work modulation is not the change in force due to a change in fiber stretch velocity (fiber damping characteristics) but the change in magnitude of the stretch due to the leg dynamics in the perturbed conditions. Our results confirm previous findings that muscle stiffness is activity-dependent and show that also damping characteristics are activity-dependent. These results indicate that neural control could tune the preflex properties of muscles in expectation of ground conditions leading to previously inexplicable neuromuscular adaptation speeds.