We aimed to determine the impact of precursor pool dilution on the assessment of postprandial myofibrillar protein synthesis rates (MPS). A Holstein dairy cow was infused with large amounts of L-[1-13C]phenylalanine and L-[1-13C]leucine and the milk was collected and fractionated. The enrichment levels in the casein were 38.7 and 9.3 MPE, respectively. In a subsequent human experiment, 11 older men (age: 71+/-1 y, BMI: 26+/-0.1 kgm-2) received a primed constant infusion of L-[ring-2H5]phenylalanine and L-[1-13C]leucine. Blood and muscle samples were collected before and after the ingestion of 20 g doubly-labeled casein to assess postprandial MPS based on the 1.) constant tracer infusion of L-[ring-2H5]phenylalanine, 2.) ingestion of intrinsically L-[1-13C]phenylalanine labeled casein, 3.) constant infusion of L-[1-13C]leucine in combination with the ingestion of intrinsically L-[1-13C]leucine labeled casein. Postprandial MPS was increased (P<0.05) after protein ingestion (~70% above postabsorptive values) based on the L-[1-13C]leucine tracer. There was no significant stimulation of postprandial MPS (~27% above postabsorptive values) when the calculated FSR was based on the L-[ring-2H5]phenylalanine (P=0.2). Comparisons of postprandial MPS based on the primed continuous infusion of L-[1-13C]leucine or the ingestion of intrinsically L-[1-13C]phenylalanine labeled casein protein demonstrated differences when compared to the primed continuous infusion of L-[ring-2H5]phenylalanine (P>0.05). Our findings confirm that the postprandial MPS assessed using the primed continuous tracer infusion approach may differ if tracer steady-state conditions in the precursor pools are perturbed. The use of intrinsically doubly-labeled protein provides a method to study the metabolic fate of the ingested protein and the subsequent postprandial MPS response.