Short-term high-fat diets may be a possible strategy to optimize the maximal fat oxidation (MFO) with the aim of increasing the contribution of fat to whole-body metabolism during prolonged exercise in order to preserve the glycogen stores and potentially postpone the onset of fatigue and improve performance1. Short-term dietary intervention may change the utilization of exo- and endogenous fat and carbohydrate sources and may influence muscle genes encoding proteins involved in lipid and carbohydrate metabolism underlying the changes in substrate utilization2. Yet, the effect of a short-term dietary intervention on MFO and the relation between changes in MFO and changes in lipid and carbohydrate metabolism-associated mRNAs following a short-term dietary intervention has not previously been examined. Therefore, this study aimed to investigate how MFO and the relative intensity that elicits MFO (Fatmax) were affected by a dietary intervention with marked changes in the distribution of dietary fat and carbohydrate intake. Moreover, it was investigated if concurrent changes in mRNAs encoding proteins involved in lipid and carbohydrate metabolism in skeletal muscle could be a contributing mechanism to the expected changes in MFO. Forty moderately trained men with a maximal oxygen uptake (VO2max) of 56.2±1.2 ml/kg/min (mean ±SEM) were allocated by stratified randomization to a 3-day isocaloric high-fat diet (HiFat) (65 E% fat, 20 E% carbohydrate, and 15 E% protein) (n=20) or high-carbohydrate diet (HiCho) (15 E% fat, 70 E% carbohydrate, and 15 E% protein) (n=20). Before and after the dietary intervention the participants underwent two identical test days in a fasted state, where a muscle biopsy was obtained and a Fatmax-test and VO2max-test were performed in order to determine MFO and Fatmax3. Real time PCR was applied to quantify changes in the mRNAs of interest, and PCR-data were normalized to the amount of total ssDNA in each sample. Mixed linear models were applied to determine possible interactions (group*time) of the dependent variables and pairwise comparisons were used to locate the differences within- and between groups. P-values 0.001) and 37±2% to 44±2% (p>0.001), respectively, in response to HiFat and decreased from 0.36±0.04 g/min to 0.28±0.06 g/min (p>0.001) and 37±2% to 33±2% (p>0.001), respectively, after HiCho. Lipid transporter cluster of differentiation 36 (CD36) and uncoupling protein-3 (UCP3) mRNA content were increased by 3.1 and 1.7-fold (p=0.018, p=0.046), respectively, after HiFat. Perilipin-5 (PLIN5) mRNA content was greater after HiFat compared to after HiCho (p=0.014), and hexokinase II (HKII) mRNA content was increased by 3.2-fold after HiCho (p=0.032). No changes were found in the mRNA content of hormone sensitive lipase(HSL), glucose transporter type 4 (GLUT4), pyruvate dehydrogenase kinase 4 (PDK4), nucleotide translocator-1 (ANT1), beta-hydroxyacyl-CoA dehydrogenase (ß-HAD), or carnitine palmitoyltransferase I  (CPT-1). These findings demonstrate that a 3-day dietary intervention affects the MFO and Fatmax and that concurrent changes in mRNAs encoding proteins related to lipid and carbohydrate metabolism in the skeletal muscle could be a potential contributing mechanism to the changes observed in MFO after HiFat and HiCho.