Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and there is great interest in understanding the potential role of alterations in mitochondrial metabolism in its pathogenesis. To address this question, we assessed rates of hepatic mitochondrial oxidation in subjects with and without NAFLD by monitoring the rate of ¹³C labeling in hepatic [5-¹³C]glutamate and [1-¹³C]glutamate by ¹³C MRS during an infusion of [1-¹³C]acetate. We found that rates of hepatic mitochondrial oxidation were similar between NAFLD and control subjects. We also assessed rates of hepatic pyruvate cycling during an infusion of [3-¹³C]lactate by monitoring the ¹³C label in hepatic [2-¹³C]alanine and [2-¹³C]glutamate and found that this flux was also similar between groups and more than 10-fold lower than previously reported. Contrary to previous studies, we show that hepatic mitochondrial oxidation and pyruvate cycling are not altered in NAFLD and do not account for the hepatic fat accumulation.