The present study examined the effect of 14 days of exposure to microgravity during the Spacelab Life Sciences-2 (SLS-2) space shuttle mission on the myosin heavy-chain (MHC) content, fibre size and type distributions and metabolic properties of rat diaphragm. Five adult male Sprague-Dawley rats were exposed to 14 days of microgravity (SF, spaceflight) and compared to five ground-based controls (C). Immunohistochemical analyses using isoform-specific anti-MHC monoclonal antibodies revealed that 14 days of SF did not alter the proportions of type-I, -IIA, -IID/X or -IIB fibres within the crural, sternal or lateral costal regions of the diaphragm; the electrophoretically quantified MHC-isoform contents also remained unchanged. In contrast, the medial gastrocnemius (MG) and tibialis anterior (TA) muscles displayed slow-to-fast fibre type transitions: within the MG the proportion of type-IID/X fibres was reduced by 59% (P<0.04) and corresponded to a 51% increase (P<0.03) in type-IIB fibres. Within the TA, the sum of type-IID/X+IIB fibres was elevated by 24% (P<0.02) at the expense of the slower type-IIA fibres, which decreased by 33% (P<0.04). Electrophoretic analyses yielded qualitatively similar patterns of transformation. SF did not induce atrophic changes within the diaphragm, MG or TA. Succinate dehydrogenase activity remained unchanged in the crural diaphragm (P>0.96) but was 34% lower (P<0.0001) in the TA. We conclude that 14 days of SF did not alter structural or metabolic factors that are known to underlie functional properties of the diaphragm. The findings of the present study show that 14 days of SF does not induce deleterious adaptive changes in the rat diaphragm that occur in hindlimb muscles.