Metabolic inflexibility is an emerging physiological marker indicative of metabolic dysfunction and associated with type 2 diabetes (T2D) and obesity. Exercise is a potent stimulus to improve metabolic health, however, not much is known about the acute effects of exercise on metabolic flexibility (METFLEX). The purpose of this dissertation was study the time-course of diet and exercise and its effects on METFLEX. Three studies were conducted to investigate molecular and clinical aspects of diet and exercise and how these stimuli may alter the metabolic response to fuel. The first study was conducted on obesity-prone, Osborne-Mendel (OM) and obesity-resistant S5B/Pl (S5B) rats. This experiment investigated inherent differences in EE, metabolic rate, METFLEX and skeletal muscle markers of metabolism, lipid storage and lipid oxidation between OM and S5B, as well as HFD-induced strain differences in these parameters. The consumption of a HFD in S5B rats increased metabolic rate and EE and decreased metabolic rate and EE in OM rats. These strain differences were not due to the differences in activity or food intake. These results suggest that HFD-induced differences in metabolic rate and EE may be mediated by HFD-induced differences in pAMPK and PPAR$γ$ expression and lipid accumulation in the gastrocnemius muscle of OM and S5B rats. The second study was designed to investigate the effect of acute aerobic exercise at an intensity that maximizes the rate of fat oxidation (FATMAX) on glucose tolerance, insulin action, and METFLEX compared to acute aerobic exercise at lactate threshold (OBLA) resulting in greater carbohydrate (CHO) oxidation. OBLA exercise appears to have deleterious effects on oral glucose tolerance and metabolic flexibility acutely, however, FM exercise does not confer improved METFLEX. These results suggest that predominate substrate utilization does not promote improved glucose tolerance and metabolic flexibility in young overweight men. The third and final study of this dissertation was designed to investigate the immediate and extended of a single bout of high intensity interval exercise (HIIE) on METFLEX and mitochondrial function. Participants were recruited with a family history (FmHx) of T2D (n=8) and without a family history of T2D (n=8). The extended effects (48 hours) work via the reduction fat oxidation by increasing resting fat oxidation rate and improving the suppression of fat oxidation in response to a mixed meal.