In a unilaterally denervated diaphragm, atrophy of fast-twitch glycolytic (FG) fibers, but not of fast-twitch oxidative glycolytic (FOG) or slow-twitch oxidative (SO) fibers, occurs. The inhibition of atrophy in FOG and SO fibers may be due to the effects of stretch stimuli caused by respiratory contraction of the contralateral diaphragm. We examined the effects of exercise (treadmill running) on the histochemical and biochemical properties of muscle fibers of the costal diaphragm in three groups. Eighteen rats were distributed in (i) a denervation + treadmill running (Dn ex) group, (ii) a denervation (Dn) group, and (iii) a control (Ct) group. After 4 weeks of running, we measured the expression of myosin heavy chain (MHC) isoforms and cross-sectional area of the diaphragm muscle. The expression of MHC 1 and MHC 2a increased 4 weeks after denervation. In contrast, the expression of MHC 2d decreased significantly. After 4 weeks of running, the cross-sectional area of FG fibers increased significantly (85.5%; P < 0.05) compared with that in the Dn group. We suggest that exercise enhances the morphological properties and myosin heavy chain isoforms of the denervated diaphragm. This exercise-mediated adaptation may be associated with the improvement of FG fibers in the denervated diaphragm.