The diffusive properties of adjacent muscles at rest were evaluated in male (n = 12) and female (n = 12) subjects using diffusion tensor imaging (DTI). The principle, second and third eigenvalues, trace of the diffusion tensor [Tr(D)], and two anisotropic parameters, ellipsoid eccentricity (e) and fractional anisotropy (FA), of various muscles in the human calf were calculated from the diffusion tensor. Seven muscles were investigated in this study from images acquired of the left calf: the soleus, lateral gastrocnemius, medial gastrocnemius, posterior tibialis, anterior tibialis, extensor digitorum longus and peroneus longus. A mathematical model was also derived that relates the eigenvalues of the diffusion tensor to the muscle fiber volume fraction, which is defined as the volume of muscle fibers within a well-defined arbitrary muscle volume. Females on average had higher eigenvalues and Tr(D) compared with males, with the majority of muscles being statistically different between the sexes. In contrast, males on average had higher e and FA than females, with the large plantar flexors--soleus, lateral gastrocnemius, and medial gastrocnemius--producing statistically different results. The behavior of the mathematical model for variations in fiber volume fraction produced similar trends to those seen when the experimental data were fit to the model. The model predicts that a larger volume fraction of skeletal muscle in males is devoted to fibers than in females, but the true underlying source of the gender discrepancy remains unclear. Although the model does not fully account for other transport processes, it does provide some insight into the limiting factors that affect the diffusion of water in skeletal muscle measured by DTI.