Hyperthermia was shown to induce oxidative stress by uncoupling mitochondrial respiratory chain and to reduce superoxide dismutase (SOD) activity in muscles. Reactive carbonyl groups, malondialdehyde (MDA)-protein adducts, 3-nitrotyrosine immunoreactivity, Mn-SOD, and catalase were detected using immunoblotting in rat diaphragm specimens and homogenates thawed at room temperature (after previous storage at -80 degrees C) for 5, 15, 30, and 60 min, and 3, 6, and 24h to be subsequently and immediately stored at -80 degrees C. Mn-SOD activity was also measured in all muscles. Both total protein carbonylation (reactive carbonyl groups and MDA-protein adducts) and nitration were significantly increased over time, reaching their peaks in the diaphragms of the 60- and 15-min groups, respectively. Mn-SOD expression and activity were significantly reduced over time, while catalase expression showed no significant variation. Protein oxidation was significantly increased in the rat diaphragms exposed to freezing-thawing cycles of different time lengths, while Mn-SOD was substantially reduced in all muscles.