Spearman’s correlation coefficent was employed to assess whether the abundances of two taxa were independent or associated. Obesity is closely linked with insulin resistance, and increasing evidence suggests that reactive oxygen species generated in muscle mitochondria may impair insulin signalling in animal and cellular models. These studies have also shown that over – expression of muscle-specific antioxidant enzymes, or treatment with the mitochondrial superoxide dismutase mimetics and mitochondria-specific free radical scavengers, protects rodents from developing insulin resistance following high fat overfeeding. Whether sustained high fat overfeeding will elevate these markers in non-obese humans is not yet clear, although a single high fat meal increases mitochondrial ROS emission in lean and obese humans. Moreover, both systemic markers of oxidative stress and ROS production in skeletal muscle mitochondria are reported to be elevated in human obesity. A reduction in skeletal muscle mitochondrial number and/or maximal oxidative capacity is also reported in human obesity, aging and type 2 diabetes and is postulated to be causal in the development of obesity-associated insulin resistance. The mitochondrial dysfunction hypothesis of insulin resistance has arisen SCH772984 mainly from studies showing reduced expression of genes involved in mitochondrial biogenesis or reduced ATP production in healthy relatives of type 2 diabetes individuals. Reduced expression of genes involved in mitochondrial biogenesis is also observed following isocaloric high fat diet, or following prolonged lipid infusion with the parallel induction of peripheral insulin resistance in healthy humans. However, other studies have shown that mitochondrial dysfunction is not a prerequisite for insulin resistance in humans. Rodents that are fed a high fat diet for 4–20 weeks have increases in the more functional measures of skeletal muscle oxidative capacity, despite developing insulin resistance and diabetes. Together, these findings challenge the role of mitochondrial dysfunction as a primary factor in the development of insulin resistance. We, and others, have previously shown that short term overfeeding decreases the glucose infusion rate necessary to maintain euglycemia during a hyperinsulinemic-euglycemic clamp. In this study, we focused on factors in skeletal muscle that may contribute to the insulin resistance that was observed during overfeeding. The specific aims were to determine the effects of 3 and 28 days of overfeeding on skeletal muscle markers of oxidative stress, and mitochondrial content and function. We hypothesized that overfeeding would increase oxidative stress and this would be associated with a reduction in markers of mitochondrial content and function. Short term overfeeding reduces insulin sensitivity in healthy non-obese individuals, however the mechanisms underlying this are unclear. In this study, we report that whilst the reduction in insulin sensitivity following overfeeding was modest, it occurred without a reduction in any of the markers of mitochondrial content and function examined. However, we observed that systemic and skeletal muscle markers of oxidative stress were increased, and therefore may have contributed to the insulin resistance observed. The role of ROS in mediating insulin resistance is debated.