Continual intake of high-fat foods, coupled with limited physical activity, can lead to metabolic inflexibility. Eventually, this may lead to significant health issues such as obesity, insulin resistance, cardiovascular disease, and other chronic diseases. Metabolic flexibility of human skeletal muscles is influenced by changes to mitochondrial, nuclear, and cytosolic proteins, in part as a result of posttranslational modifications (PTMs). O-linked B-D-N-acetylglucosamine, known as O-GlcNAc, has recently been identified as an important posttranslational modification that responds to nutrient sensing and cellular stress. Unlike other PTMs, O-GlcNAc has only two cycling enzymes. Because of its novelty, little research has been performed on the role of O-GlcNAc in human skeletal muscle and metabolic flexibility. The purpose of the current study was to establish the effects of a 5-day high-fat diet on skeletal muscle O-GlcNAcylation. In the proposed study, 13 non-obese, sedentary, college-aged males consumed a controlled diet for two weeks followed by a high-fat diet composed of 55% fat, 30% carbohydrate, and 15% protein. Muscle biopsies were taken from the vastus lateralis both fasted and four hours after a high-fat meal, following both the control diet and the high-fat diet. Western blot analysis was used to assess global O-GlcNAc and protein concentrations of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in whole-homogenates and isolated mitochondria from skeletal muscle. Results were analyzed using independent, two-tailed t-tests and 2-way ANOVA analysis with repeated measures and Bonferroni corrections; a p-value was set to α less than or equal to 0.05. It was found that O-GlcNAc and OGT levels remained stable, although fasting levels of OGA significantly decreased after the 5-day high-fat diet. It is possible that healthy individuals are capable of maintaining normal levels of O-GlcNAc and its cycling enzymes, but there is still more to learn about O-GlcNAc and its role in metabolic flexibility.