Space flight and bed rest (BR) result in losses of muscle mass and strength. Resistance training (RT) and amino acid (AA) supplementation are potential countermeasures to minimize these losses. However, it is unknown if timing of supplementation with exercise can optimize benefits, particularly with energy deficit. We examined the effect of these countermeasures on body composition, strength, and insulin levels in 31 men (ages 31-55 y) during BR (28-d) followed by active recovery (14-d). Subjects were randomly assigned to: essential AA supplementation (AA, n=7); RT with AA given 3h after training (RT, n=12); or RT with AA given 5min prior to training (AART, n=12). Energy intake was reduced by 8±6%. Mid-thigh muscle area declined with BR for the AA>RT>AART groups: (-11, -3, -4%; p=0.05). Similarly, greatest losses in lower body muscle strength were seen in the AA group (-22%). These were attenuated in the exercising groups [RT (-8%) and AART (-6%; p<0.05)]. Fat mass and mid-thigh intramuscular fat increased after BR in the AA group (+3% and +14%, respectively), and decreased in the RT (-5% and -4%) and AART groups (-1 and -5%; p=0.05). Muscle mass and strength returned toward baseline after recovery but the AA group showed the lowest re-gains. Combined resistance training with AA supplementation pre- or post-exercise attenuated the losses in muscle mass and strength by ~2/3 compared to AA supplement alone during BR and energy deficit. These data support the efficacy of combined AA and RT as a countermeasure against muscle wasting due to low gravity.