Cardiac and skeletal muscle both respond to elevated fatty acid availability by increasing fatty acid oxidation, an effect mediated in large part by peroxisome proliferator-activated receptor-α (PPARα). We hypothesized that cardiac and skeletal muscle alter their responsiveness to fatty acids over the course of the day, allowing optimal adaptation when availability of this substrate increases. In the current study, pyruvate dehydrogenase kinase 4 (pdk4) was utilized as a representative PPARα-regulated gene. Opposing diurnal variations in pdk4 expression were observed in cardiac and skeletal muscle isolated from the ad libitum-fed rat; pdk4 expression peaked in the middle of the dark and light phases, respectively. Elevation of circulating fatty acid levels by high-fat feeding, fasting, and streptozotocin-induced diabetes increased pdk4 expression in both heart and soleus muscle. Highest levels of induction were observed during the dark phase, regardless of muscle type or intervention. Specific activation of PPARα with WY-14643 rapidly induced pdk4 expression in heart and soleus muscle. Highest levels of induction were again observed during the dark phase. The same pattern of induction was observed for the PPARα-regulated genes malonyl-CoA decarboxylase and uncoupling protein 3. Investigation into the potential mechanism(s) for these observations exposed a coordinated upregulation of transcriptional activators of the PPARα system during the night, with a concomitant downregulation of transcriptional repressors in both muscle types. In conclusion, responsiveness of cardiac and skeletal muscle to fatty acids exhibits a marked diurnal variation. These observations have important physiological and pathophysiological implications, ranging from experimental design to pharmacological treatment of patients.