Laboratory rats are considered mature at 3 months despite that musculoskeletal growth is still occurring. Changes in muscle physiological and biochemical characteristics during development from 3 months, however, are not well understood. Whole muscles and single skinned fibres from fast-twitch extensor digitorum longus (EDL) and predominantly slow-twitch soleus (SOL) muscles were examined from male Sprague-Dawley rats (3, 6, 9, 12 months). Ca2+ sensitivity of contractile apparatus decreased with age in both fast- (~ 0.04 pCa units) and slow-twitch (~ 0.07 pCa units) muscle fibres, and specific force increased (by ~ 50% and ~ 25%, respectively). Myosin heavy chain composition of EDL and SOL muscles altered to a small extent with age (decrease in MHCIIa proportion after 3 months). Glycogen content increased with age (~ 80% in EDL and 25% in SOL) and GLUT4 protein density decreased (~ 35 and 20%, respectively), whereas the glycogen-related enzymes were little changed. GAPDH protein content was relatively constant in both muscle types, but COXIV protein decreased ~ 40% in SOL muscle. Calsequestrin (CSQ) and SERCA densities remained relatively constant with age, whereas there was a progressive ~ 2-3 fold increase in CSQ-like proteins, though their role and importance remain unclear. There was also ~ 40% decrease in the density of the Na+, K+-ATPase (NKA) α1 subunit in EDL and the α2 subunit in SOL. These findings emphasise there are substantial changes in skeletal muscle function and the density of key proteins during early to mid-adulthood in rats, which need to be considered in the design and interpretation of experiments.