To fully understand the effect of scaling on skeletal muscle shortening velocity (V ₀), it is important to know which phenotypic characteristics drive the changes between species. The purpose of the current investigation was to compare the effects of body mass and femur length, as an estimate of total limb length, on V ₀ in species that cover a 100,000-fold range of body masses. Using the slack test procedure, V ₀ was determined for fibers expressing types I and IIa myosin heavy chain (MyHC) isoforms in the mouse, rat, dog, human, horse, and rhinoceros under identical experimental conditions. A significant scaling effect on V ₀ was detected when compared to body mass (type I fibers, r=0.95, p<0.01; type IIa fibers, r=0.83, p<0.05). However, the horse's V ₀ for both fiber types was faster than the human's, despite having a 5-fold greater body mass than the human. When V ₀ was scaled vs limb length, the strength of the relationships improved in fibers expressing both types I and IIa MyHC (r=0.98, p<0.001, and r=0.89, p<0.05, respectively) and scaled with the expected relationship, with the species with the shorter femur, the horse, having the faster V ₀. A similar effect can be seen with stride frequency scaling more closely with limb length than body mass. These results suggest that limb length, not body mass, is a more relevant factor driving the scaling effect on skeletal muscle shortening velocity.