Increased ROS production is common to different models of cellular insulin resistance, including those induced by TNF-a, insulin and palmitate treatments. Moreover, mitochondria-targeted Wortmannin antioxidant treatment partially preserves insulin sensitivity both in vivo and in vitro. In the present study, we observed that both urinary F2-isoprostane and skeletal muscle protein carbonyls were increased, with the latter increased as early as 3 days of overfeeding. This finding suggests that increased oxidative stress may be an early event during over-nutrition in humans. Protein carbonylation is a nonreversible modification by highly reactive aldehydes, by-products of lipid peroxidation that cause loss of function or trigger degradation of proteins with a cysteine, histidine or lysine side chain, typically enzymes. Carbonylated proteins, including the antioxidants thioredoxin, thioredoxin reductase, glutathione peroxidase, fatty acid binding protein and cytosolic and mitochondrial NADP+ -dependent isocitrate dehydrogenase isoforms, were 2–3 fold higher in adipose tissue collected from high fat-high sucrose fed mice compared to chow fed mice. We speculate that antioxidants and enzymes involved in oxidative stress and/or insulin action in skeletal muscle may also be potential targets for carbonylation and degradation during the overfeeding diet. Also, in the postprandial state, fat and carbohydrate have a differential effect on the oxidative stress response. Importantly, participants in the present study were placed on the same snacks, rich in both sugar and fat, to increase their energy intake and thus we cannot differentiate the effect of particular macronutrients on the outcomes. The two principal sites of superoxide generation in mitochondria are complexes I and III of the electron transport chain. In this study, we observed an increase in protein content of complex I, but not complex III, at day 3 of overfeeding. Superoxide leaking from the mitochondrial complexes is dismutated rapidly into hydrogen peroxide by MnSOD and Cu/ZnSOD in the mitochondrial matrix and the inter-membrane space, respectively. Consistent with this, it has previously been shown that MnSOD transgenic mice are partially protected from high fat feeding-induced insulin resistance. In the present study, we observed that MnSOD was increased transiently, possibly in an attempt to limit oxidative damage. We speculate that the lack of a sustained induction of the anti-oxidative systems, including MnSOD and UCP3 may have contributed to the increase in oxidative stress that was observed following overfeeding in this study. Previous studies have shown that high fat diet increased UCP3 protein in rodent mitochondria and isocaloric 65% fat diet increased UCP3 mRNA expression in lean, but not obese humans. However, to our knowledge protein content of UCP3 during overfeeding has not previously been investigated. Pre-diabetes and type 2 diabetes are characterized by reduced expression and protein levels of PGC1a, a master metabolic regulator of mitochondrial biogenesis. However, it is unclear whether this is a cause or consequence of insulin resistance. In the present study, the protein levels of PGC1a and the complexes of the mitochondrial electron transport chain were increased at day 3, but these returned to basal at day 28.