Every-other-day-fasting (EODF) is an intermittent fasting regime that improves insulin sensitivity and lifespan in model animals without weight loss. However, the underlying mechanisms linking the dietary perturbation and the beneficial phenotypes remain to be uncovered. Here, we have employed proteome analysis of mouse liver, a key fasting-responsive organ, to identify the key protein abundance changes occurring in response to the EODF intervention compared to ad libitum fed animals. From >6,000 proteins quantified, more than 250 proteins were significantly altered by the EODF intervention. Among the most up-regulated proteins after EODF was acyl-CoA thioesterase2 (ACOT2), which can accelerate liver fatty-acid oxidation and when up-regulated is known to have beneficial effects on whole-body metabolism. Surprisingly, alpha1-antitrypsin (SERPINA1) was the most down-regulated protein (>16-fold) after the EODF intervention. SERPINA1 function has previously been linked with lipoprotein particle metabolism in mice and humans. Given that SERPINA1 is among the 10 most abundant proteins in blood plasma and only synthesized in the liver, we performed single-shot plasma proteome analysis to quantify the top 200 most abundant proteins. This analysis showed that SERPINA1 was also down-regulated ~3-fold in plasma of EODF animals, in addition to >20 other significant protein changes. We subsequently showed that the SERPINA1 protein abundance change in liver is matched by a significant change in SERPINA1 liver mRNA abundance. HNF4A is a known regulator of the SERPINA1 promoter for induction of transcription. We have used CHIP-qPCR analysis to quantify the changes in association between HNF4A and the promoter of either SERPINA1, or a canonical HNF4A-target gene (ABCC6). This analysis showed that the association of HNF4A with both promoters was reduced >5-fold by the EODF intervention. These data suggest that HNF4A is globally regulated during EODF, although the abundance of HNF4A was not significantly altered in our liver proteome data. Therefore, we hypothesise that HNF4A is regulated by either post-translational modification and/or changes in protein-protein interactions. Immunoprecipitations of HNF4A from the liver tissue are on-going to identify significant differences in HNF4A modifications and interactions.