Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focussed primarily at the physiologic and gene expression levels, but these analyses are invariably incomplete not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24 hour (h) day/night murine cardiac proteome was assessed by 2-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ~7.8% (90/1147) of spots exhibited statistical changes at paired times across the 24h light (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles, and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance, and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.