Growing evidence supports the theory that mitochondrial dysfunction is an underlying cause of intramyocellular lipid (IMCL) accumulation and insulin resistance (IR). Here we hypothesized that high dietary fat (HF) intake could trigger changes in mitochondrial activity such that fatty acid oxidation is impaired in muscle and contributes to an elevation in intramyocellular lipid (IMCL) levels. Muscle mitochondrial activity was determined in vivo through measurement of the F₁F₀ ATP synthase flux, the terminal step in the oxidative phosphorylation process. An initial study comparing rats on normal chow diet with rats on high-fat diet revealed strong correlations between muscle ATP synthesis rates, IMCL levels and whole-body glucose tolerance. Results obtained from two latter studies showed multiphasic responses to dietary intervention. Initially, the ATP synthesis rates decreased as much as 50% within 24 hours of raising the fat content in the diet to 60% of the caloric intake. These rates eventually returned to normal values after 2 to 3 weeks on the HF regimen, seemingly to prevent further IMCL accumulation. Only beyond one month on HF diet did results consistently show ATP synthesis rates to diminish by 30 to 50% accompanied by steadily augmenting IMCL levels. Interestingly, switching back to chow diet after 3 weeks of HF feeding reversed the initial diet induced changes. Although the muscle mitochondrial system may initially offer enough compliance to counteract lipid surplus, these in vivo data suggest a vicious long-term cycle between mitochondrial dysfunction, IMCL accumulation and glucose intolerance in the rat.