Consuming 0.10-0.14g essential amino acids (EAA)/kg/dose (0.25-0.30g protein/kg/dose) maximally stimulates muscle protein synthesis (MPS) during energy balance. Whether consuming EAA beyond that amount enhances MPS and whole-body anabolism following energy deficit is unknown. The aims of this study were to determine the effects of standard and high EAA ingestion on mixed MPS and whole-body protein turnover following energy deficit. Nineteen males (mean±SD; 23±5y; 25.4±2.7kg/m2) completed a randomized, double-blind crossover study consisting of two, 5-d energy deficits (-30±4% of total energy requirements), separated by 14-d. Following each energy deficit, mixed MPS and whole-body protein synthesis (PS), breakdown (PB), and net balance (NET) were determined at rest and post-resistance exercise (RE) using primed, constant L-[2H5]-phenylalanine and L-[2H2]-tyrosine infusions. Beverages providing standard (0.1g/kg, 7.87±0.87g) or high (0.3g/kg, 23.5±2.54g) EAA were consumed post-RE. Circulating EAA were measured. Postabsorptive mixed MPS (%/h) at rest was not different (P=0.67) between treatments. Independent of EAA, postprandial mixed MPS at rest (standard EAA, 0.055±0.01; high EAA, 0.061±0.02) and post-RE (standard EAA, 0.055±0.01; high EAA, 0.065±0.02) were greater than postabsorptive mixed MPS at rest (P=0.02 and P=0.01, respectively). Change in (Δ postabsorptive) whole-body (g/180min) PS and PB was greater for high than standard EAA [mean treatment difference (95% CI), 3.4 (2.3, 4.4); P=0.001 and-15.6 (-17.8, -13.5); P=0.001, respectively]. NET was more positive for high than standard EAA [19.0 (17.3, 20.7); P=0.001]. EAA concentrations were greater in high than standard EAA (P=0.001). These data demonstrate that high compared to standard EAA ingestion enhances whole-body protein status during underfeeding. However, the effects of consuming high and standard EAA on mixed MPS are the same during energy deficit. NCT03372928, https://clinicaltrials.gov.