RATIONALE: Conventionally, myofibrillar protein synthesis is measured over time periods of hours. In clinical studies, interventions occur over weeks. Functional measures over such periods may be more representative. We aimed to develop a novel method to determine myofibrillar protein fractional synthetic rate (FSR) to estimate habitual rates, while avoiding intravenous tracer infusions. METHODS: Four healthy males were given 100â€‰g water enriched to 70 Atom % with 2H2O as a single oral bolus. Vastus-lateralis needle biopsies were performed and plasma samples collected, 3?13â€‰days post-dose. 2H enrichment in body water was measured in plasma using continuous flow isotope ratio mass spectrometry (IRMS). Myofibrillar protein was isolated from muscle biopsies and acid hydrolysed. 2H enrichment of protein-bound and plasma-free alanine was measured by gas chromatography (GC)/pyrolysis/IRMS. Myofibrillar protein FSR was calculated (% day?1). RESULTS: The tracer bolus raised the initial enrichment of body water to 1514â€‰ppm 2H excess. Water elimination followed a simple exponential. The average elimination half-time was 8.3â€‰days. Plasma alanine, labelled during de novo synthesis, followed the same elimination kinetics as water. The weighted average myofibrillar protein FSR from the four subjects was 1.38 % day?1 (range, 1.0?1.9 % day?1). CONCLUSIONS: Myofibrillar protein FSR was measured in free-living healthy individuals over 3?13â€‰days. Using a single oral 2H2O bolus, endogenous labelling of alanine occurred in a predictable manner giving estimates of synthesis comparable with published values. Furthermore, the protocol does not compromise the ability to measure other important metabolic processes such as total energy expenditure.