Macronutrient metabolism plays an essential role in the overall health of an individual. Depending on a number of variables, for example, diet, fitness level, or metabolic disease state, protein, carbohydrate and fat have varying capacities to be oxidized and balanced. Further, when analyzing the oxidation of carbohydrate and fat in the skeletal muscle specifically, carbohydrate balance happens quite rapidly, while fat balance does not. The ability of skeletal muscle to adapt and respond to various nutrient states is critical to maintaining healthy metabolic function. Habitual high fat intake has been associated with reduced oxidative capacity, insulin resistance, increased gut permeability, inflammation, and other risk factors often preceding metabolic disease states. The disruption of gut function leads to gut permeability and increases endotoxins released into circulation. Endotoxins have been shown to play an important role in obesity-related whole body and tissue specific metabolic perturbations. Each of these disrupted metabolic processes is known to associate with obesity, metabolic syndrome and diabetes. To date, limited research has investigated the role of high fat diet on skeletal muscle substrate oxidation and its relationship to gut permeability and endotoxins. The purpose of this study was to determine the effects of an acute, five-day, isocaloric high fat diet (HFD) on skeletal muscle substrate metabolism in healthy non-obese humans. An additional purpose was to determine the effects of a HFD on gut permeability and blood endotoxins on healthy, non-obese, sedentary humans. Thirteen college age males were fed a control diet for two weeks, followed by five days of an isocaloric HFD. To assess the effects of a HFD on skeletal muscle metabolic adaptability and postprandial endotoxin levels, subjects underwent a high fat meal challenge before and after a HFD. Muscle biopsies were obtained; blood was collected; insulin sensitivity was assessed via intravenous glucose tolerance test; and intestinal permeability was assessed via the four-sugar probe test before and after the HFD. Postprandial glucose oxidation and fatty acid oxidation in skeletal muscle increased before the HFD intervention but was decreased after. Skeletal muscle in vitro assay of metabolic flexibility was significantly blunted following the HFD. Insulin sensitivity and intestinal permeability were not affected by HFD, but fasting endotoxin was significantly higher following the HFD. These findings demonstrate that in young, healthy males, following five days of an isocaloric high fat diet, skeletal muscle metabolic adaptation is robust. Additionally, increased fasting endotoxin independent of gut permeability changes are potentially a contributor to the inflammatory state that disrupts substrate oxidation. These findings suggest that even short-term changes in dietary fat consumption have profound effects on skeletal muscle substrate metabolism and fasting endotoxin levels, independent of positive energy balance and whole-body insulin sensitivity.