Energy deficit has shaped human evolution, and represents a potent physiological stressor associated with improvements in lifespan and healthspan in several species. Preserving locomotive capacity has likely been essential for food procurement and survival from starvation during the hunter-gatherer period. However, we have very limited understanding of the effect of energy deficit on skeletal muscle, a key tissue for locomotion and metabolic health. Here we show that a 5-day 78% reduction in energy availability with concomitant exercise in healthy men leads to 2.1 ±0.8 kg loss of fat free mass and 0.8 ±0.6 kg of fat mass; increases fat oxidation at rest and during exercise, and induces metabolic and endocrine shifts associated to energy preservation, concomitant with a profound modulation of skeletal muscle phenotype. We used stable isotope (deuterium oxide) labelling and peptide mass spectrometry to investigate the abundance and turnover rates of individual proteins. Abundance (1469 proteins) and absolute synthesis rates (736 proteins) show a shift towards an oxidative phenotype, and cytoskeleton and extracellular matrix remodelling during energy deficit compared to energy balance. Mitochondrial components: TCA, electron transport chain and beta-oxidation, are prominently represented amongst the proteins that increase in abundance and synthesis rate, as well as proteins associated to mitochondrial proteostasis, remodelling and quality-control such as BDH1 and LONP1. The changes in muscle metabolic pathways occurred alongside a reduction in extracellular matrix proteins, which may oppose the age-related increases in fibrotic tissue in muscle. Our results suggest that muscle metabolic pathways are not only preserved but positively affected during periods of low energy availability and physical demands.