To investigate the role of hepatocyte genes in fasting metabolism, it is essential to analyze their expression across the entire 24-h circadian cycle. Here, we present a protocol to evaluate fasting metabolism and its relationship to the core circadian clock in mice. We describe steps for time-course fasting/feeding experiment setup and performing staggered fasting and refeeding for 24 h. We then detail procedures for evaluating oxidative substrate selection and rescuing defective oxidative metabolism through FGF21 and CPI-613 injections. For complete details on the use and execution of this protocol, please refer to Sun et al.