Updated project metadata. Fasting is a common dietary intervention known for its protective effects against metabolic and cardiovascular diseases. While its effects are mostly systemic, understanding tissue-specific changes in the heart is crucial for the identification of the mechanisms underlying fasting-induced cardioprotection. In this study, we performed a proteomic analysis to elucidate the proteome of the fasting heart. Our investigation identified a total of 4,652 proteins, with 127 exhibiting down-regulation and 118 showing up-regulation after fasting. Annotation analysis highlighted significant changes in processes such as lipid metabolism, the peroxisome pathway, and reactive oxygen species metabolism. Notably, the HIF-1 signaling pathway emerged as one of the focal points, with five affected proteins identified. Further experiments demonstrated down-regulation of HIF-1α at both transcript and protein levels. Intriguingly, while gene expression of Egln3 decreased, its protein product PHD3 remained unaffected by fasting. These findings underscore the regulation of the well-established cardioprotective HIF-1 signaling within the heart during 3-day fasting.