To date, few studies have characterized the influence of energy deprivation on direct measures of skeletal muscle protein turnover. In this investigation, we characterized the effect of an acute, moderate energy deficit (10 d) on mixed muscle fractional synthetic rate (FSR) and associated intracellular signaling proteins in physically active adults. Eight men and 4 women participated in a 20-d, 2-phase diet intervention study: weight maintenance (WM) and energy deficient (ED; approximately 80% of estimated energy requirements). Dietary protein (1.5 g x kg(-1) x d(-1)) and fat (approximately 30% of total energy) were constant for WM and ED. FSR and intracellular signaling proteins were measured on d 10 of both interventions using a primed, constant infusion of [(2)H(5)]-phenylalanine and Western blotting techniques, respectively. Participants lost approximately 1 kg body weight during ED (P < 0.0001). FSR was reduced approximately 19% (P < 0.05) for ED (0.06 +/- 0.01%/h) compared with WM (0.074 +/- 0.01%/h). Protein kinase B and eukaryotic initiation factor 4E binding protein 1 phosphorylation were lower (P < 0.05) during ED compared with WM. AMP activated protein kinase phosphorylation decreased (P < 0.05) over time regardless of energy status. These findings show that FSR and associated synthetic intracellular signaling proteins are downregulated in response to an acute, moderate energy deficit in physically active adults and provide a basis for future studies assessing the impact of prolonged, and perhaps more severe, energy restriction on skeletal muscle protein turnover.