Every-other-day-fasting (EODF) is an effective intervention for treatment of metabolic disease including improvements in liver health. But how the liver proteome is reprogrammed by EODF is currently unknown. Here, we employed EODF in mice and multi-Omics analysis to identify key regulated pathways. Many changes in the liver proteome and metabolome were distinct in EODF animals and not present after a single fasting bout. Key among these was the simultaneous induction by EODF of de novo lipogenesis and fatty-acid oxidation enzymes. Together with activation of oxidative stress response pathways, these changes would contribute to the improvements in glucose tolerance and lifespan after EODF. Enrichment analysis showed unexpected downregulation of HNF4A targets by EODF and we confirmed inhibition of HNF4A function. Suppressed HNF4A targets include bile synthetic enzymes and secreted proteins such as a1-antitrypsin and inflammatory factors, which reflect known EODF phenotypes and were confirmed by plasma proteome analysis. Interactive online access is provided to this data resource (larancelab.com/eodf), which provides a global view of fasting-induced mechanisms in mice.