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Background Diffusion-weighted imaging (DWI) and diffusion-tensor imaging (DTI) can be used to evaluate changes that accompany skeletal muscle contraction. Purpose To investigate whether jaw opening or closure affect the diffusion parameters of the masseter muscles (MMs). Material and Methods Eleven healthy volunteers were evaluated. Diffusion-tensor images were acquired to obtain the primary (λ1), secondary (λ2), and tertiary eigenvalues (λ3). We estimated these parameters at three different locations: at the level of the mandibular notch for the superior site, the level of the mandibular foramen for the middle site, and the root apex of the mandibular molars for the inferior site. Results Both λ2 and λ3 during jaw opening were significantly lower than that at rest at the superior ( P = 0.006, P < 0.0001, respectively) and middle site ( P = 0.004, P = 0.0001, respectively); however, the change in λ1 was not significant. At the lower site, no parameter was significantly different at rest and during jaw opening. There was no significant difference in T2 between at rest (40.3 ± 4.4 ms) and during jaw opening (39.2 ± 2.7 ms; P = 0.12). The changes induced by jaw closure were marked at the inferior site. In the middle and inferior sites, the three eigenvalues were increased by jaw closure, and the changes in λ1 ( P = 0.0145, P = 0.0107, respectively) and λ2 ( P = 0.0003, P = 0.0001) were significant (especially λ2). Conclusion The eigenvalues for diffusion of the MM were sensitive to jaw position. The recruitment of muscle fibers, specific to jaw position, reflects the differences in changes in muscle diffusion parameters.