OBJECTIVES: Short-term energy deficit reduces acute measures of mixed muscle protein synthesis (MPS) and suppresses the hypothalamic-pituitary axis and endogenous testosterone synthesis. We hypothesized that testosterone supplementation could mitigate the effects of energy deficit on MPS. We conducted a randomized, double-blind, placebo-controlled trial to determine the effects of 28 days of tightly-controlled severe energy deficit (deficit 55% of total energy requirements) on measures of mixed-MPS and proteome-wide protein dynamics in non-obese men either given 200 mg testosterone enanthate (Testosterone, n = 24) or placebo (Placebo, n = 26) injections per week. METHODS: Participants received daily aliquots of deuterated water ((2)H(2)O) for 42 consecutive days (14-d weight maintenance period followed by 28-d energy deficit). Muscle biopsies were collected at rest in a fasted state at the end of the weight maintenance phase (PRE) and at the middle (MID) and end (POST) of the 28-d energy deficit. Mixed-MPS and proteome-wide protein fractional synthesis rates (FSR) were quantified. Changes over time and differences between Testosterone and Placebo were determined for mixed-MPS, and cross-sectional comparisons between Testosterone and Placebo were performed at MID and POST for proteome dynamics. RESULTS: In both Testosterone and Placebo, mixed-MPS were 40% and 33% lower (P