Duchenne Muscular Dystrophy (DMD) is a neuromuscular disorder characterized by progressive muscle weakness as well as progressive muscle fat replacement. Absence of functional dystrophin in muscles is believed to cause membrane instability, making the sarcolemma susceptible to damage when subjected to mechanical stress. However, the process leading from dystrophin-absence to muscle degeneration, and ultimately fat replacement, is still not well understood. Therefore, several hypotheses currently co-exist. Interestingly, muscles of DMD patients are not equally affected, and a more or less consistent successive pattern of muscle fat replacement exists in the lower extremities. The aim of this study was to investigate a possible mechanical role in explaining differential muscle fat replacement in the legs of DMD patients. Based on a published preliminary study, it was hypothesized that eccentric contraction, during which a muscle generates force while lengthening, is associated to muscle fat replacement during gait. 3D gait analysis data of a small cross-sectional sample of typically developing children was used to predict muscle contributions during gait in OpenSim. In one analysis, negative work, the time-integral of force multiplied by lengthening velocity, was normalized to account for differences in physiological cross-sectional area (PCSA) and fiber length, and was subsequently tested for a relation with muscle fat replacement. Furthermore, since muscle architecture parameters PCSA and fiber length are associated to muscle fat replacement, separate effects of total negative work and muscle volume (PCSA*fiber length) on muscle fat replacement were also analyzed. Normalized negative work was significantly associated with muscle fat replacement, where upper leg muscles generally showed higher fat fractions than lower leg muscles. Furthermore, both the effect of total negative work as well as muscle volume on muscle fat replacement were significant. The effect of total negative work on muscle fat replacement was stronger if muscle volume was smaller. In contrast, normalized and total positive work (i.e., concentric contraction) was not significantly associated with muscle fat replacement. Eccentric contraction during gait helps explain the differential progression of muscle fat replacement seen among leg muscles of DMD patients. The results of this study generate more insight regarding a mechanical role in the pathophysiology of DMD, and may potentially help in generating or improving therapies that focus on prolonged muscle function preservation.