Stable isotopically-labelled amino acids (AA) are a crucial tool for developing our understanding of the control of human muscle protein synthesis (MPS) in health, ageing and catabolic disease(s) (1). Typically, intravenous (I.V) infusions of heavy carbon/ nitrogen/ hydrogen AA-tracers alongside tissue [muscle] sampling and mass spectrometric (MS) analyses permit calculation of MPS as a fractional rate of protein synthesis (FSR). Yet, despite providing highly accurate dynamic readouts of musculoskeletal metabolism, these traditional AA-tracer methodologies are subject to limitations. These approaches, require I.V infusions and venous cannulation(s), which can be challenging in some populations (e.g. frail elderly), and are costly. Recently, we developed less invasive methods and showed that oral provision of deuterium oxide (D2O) tracer was efficacious for quantifying MPS over 2-8 days (2). The sensitivity of our approach to quantify short-term MPS (i.e. over hours) remained unknown. Here, we hypothesized that our newly developed precision MS approaches coupled to appropriate D2O dosing strategies would permit accurate quantification of MPS over periods of hours. We recruited nine males (24±3y, BMI: 25±3kg.m-², ±SD) into an internally controlled comparison of D2O vs. AA tracers. Having orally consumed 400 ml D2O ~18 h earlier, on the study day subjects received I.V infusions of L-[ring-13C6]-phenylalanine (0.3 mg.kg-1 prime, 0.6 mg.kg.h-1, continuous) to permit bi-tracer quantification of MPS under: i) basal [postabsorptive] and, ii) stimulated [postprandial] conditions i.e. consumption of 20g EAA. Muscle biopsies were taken from m. vastus lateralis at 0h, 3h and 6h (i.e. 0-3 h, basal MPS, and 3-6 h, impacts of EAA anabolic stimulus), with blood and saliva samples being taken regularly throughout the study. The FSR of myofibrillar protein fractions were quantified by GC-C-IRMS (13C6) and GC-pyrolysis-IRMS (D2O) techniques, using intracellular phenylalanine and body water as surrogate precursors; respectively. Postabsorptive rates of MPS were equivalent between tracers i.e. D2O: 0.050±0.007 and 13C6: 0.065±0.004 %.h-1 (±SEM). Similarly, increases in MPS following EAA consumption were both significant (P<0.05, two-way ANOVA) and indistinguishable between tracers: 0.088±0.008 and 0.089±0.006 %.h-1 using D2O and 13C6; respectively. Moreover, despite moderate intra-individual tracer-dependent differences in FSR (NB. not different from other cross tracer comparisons), there was a significant Pearsons correlation between D2O and ¹³C6 phenylalanine derived FSR (r=0.438, P=0.035 one-tailed), whilst Bland-Altman plots showed only a small mean bias of 0.0083 between the two methods. As such, we have developed, and validated, a less invasive D2O method for the acute quantification of MPS.