In order to increase our understanding of diabetes-related muscle weakness, we carried out a mass spectrometry-based proteomic analysis of skeletal muscle preparations from the Goto-Kakizaki rat model of type-2 diabetes. Fluorescence difference in-gel electrophoresis was performed to determine potential differences in the global protein expression profile of muscle extracts. Besides changes in contractile proteins and metabolic enzymes, the abundance of the small stress proteins αB-crystallin and Hsp27 was significantly increased. The up-regulation of the low-molecular-mass heat shock protein Hsp27 was confirmed by an alternative fluorescent staining method of two-dimensional gels and immunoblotting. The observed protein alterations in the cellular stress response, distinct metabolic pathways, regulatory mechanisms and the contractile apparatus might be directly or indirectly associated with peripheral resistance to insulin signalling, making these newly identified muscle proteins potential biomarkers of type-2 diabetes. Increased levels of molecular chaperones suggest considerably enhanced cellular stress levels in diabetic muscle fibres.