Intermittent fasting (IF) is a beneficial dietary treatment for obesity which improves metabolic health independent of weight loss. Recently, we completed the first proteomic analysis of IF in male and female mice, which uncovered a sexually dimorphic response to IF in the liver. This response included a divergence in fatty acid metabolism between the sexes and an EODF induced potent interferon-a (IFNα) signalling in female mice. Male and female C57B/6J mice were subject to a 2-week every-other-day fasting (EODF) model with chow and compared against an ad libitum control. Metabolic health was assessed throughout the model, tissues were snap frozen on LN2 and submitted to a standard trypsin digest and stage-tip cleanup. Samples were analysed using conventional DIA proteomics on a QExactive HFX-fusion type instrument. Data was analysed using the DIA-NN search engine and subject to statistical analysis by two-way ANOVA. These analyses consistently identified >4,500 proteins across the cohort. Animals of both sexes had increased fatty acid catabolism, but this change was much greater in females whereas males had greater increases in fatty acid synthesis. Interestingly, females had increased interferon-alpha signalling after IF and increased abundance of downstream IFNα targets, whereas males had minimal change. Then we applied IF to castrated mice, which showed testosterone signalling represses IFNα pathway induction in the liver after IF. Next, mice that had one of the IFNα receptors knocked out (IFNAR1) before IF, where these animals produced no IFNα signalling response demonstrating that receptor engagement is necessary. We hypothesis that IFNα signalling is tied to lipid biosynthetic flux and as this pathway is downregulated during the fasting period it engages IFNα through the STING pathway. This work highlights an interesting intervention that improves metabolic health in the absence of weight loss and is a novel opportunity for metabolic disease treatment.