Technical Abstract: Multiple extra-cardiac stimuli, such as workload and circulating nutrients (e.g., fatty acids), known to influence myocardial metabolism and contractile function exhibit marked circadian rhythms. The aim of the present study was to investigate whether the rat heart exhibits circadian rhythms in its responsiveness to changes in workload and/or fatty acid (oleate) availability. Thus, hearts were isolated from male Wistar rats (housed in a 12h:12h light:dark cycle; lights on at 9AM) at 9AM, 3PM, 9PM, and 3AM, and perfused in the working mode ex vivo with 5mM glucose, plus either 0.4mM or 0.8mM oleate. Following 20 min perfusion at normal workload (i.e., 100cm H2O afterload), hearts were challenged with increased workload (140cm H2O afterload plus 1µM epinephrine). In the presence of 0.4mM oleate, myocardial metabolism exhibited a marked circadian rhythm, with decreased rates of glucose oxidation, increased rates of lactate release, decreased glycogenolysis capacity, and increased channeling of oleate into non-oxidative pathways during the light phase. Rat hearts also exhibited a modest circadian rhythm in responsiveness to the workload challenge, when perfused in the presence of 0.4mM oleate, with increased myocardial oxygen consumption at the dark-to-light phase transition. However, rat hearts perfused in the presence of 0.8mM oleate exhibited a markedly blunted contractile function response to the workload challenge during the light phase. In conclusion, these studies expose marked circadian rhythmicities in myocardial oxidative and non-oxidative metabolism, as well as responsiveness of the rat heart to changes in workload and fatty acid availability