Abstract The goal of this investigation was to assess the influence of tracer selection on mixed muscle fractional synthesis rate (FSR) at rest and postexercise during amino acid infusion in multiple human skeletal muscles. Fractional synthesis rate was measured before and 24 hours after 45 minutes of running using simultaneous infusion of [ 2H 5]-phenylalanine (Phe) and [ 2H 3]-leucine (Leu) coupled with muscle biopsies from the vastus lateralis and soleus in aerobically trained men (n = 8; age, 26 ± 2 years). Mixed muscle protein FSR was analyzed by gas chromatography–mass spectrometry combined with a standard curve using the enriched muscle tissue fluid as the precursor pool. To control for potential analytical differences between tracers, all samples and standards for both tracers were matched for m + 0 abundance. Tracer selection did not influence resting FSR for the vastus lateralis or soleus ( P > .05). Fractional synthesis rate measured 24 hours postexercise was higher ( P < .05) compared with rate at rest and was similar between tracers for the vastus lateralis (Phe, 0.110% ± 0.010%·h −1; Leu, 0.109% ± 0.005%·h −1) and soleus (Phe, 0.123% ± 0.008%·h −1; Leu, 0.122% ± 0.005%·h −1). These data demonstrate that tracer selection does not influence the assessment of resting or postexercise FSR, thereby supporting the use of both [ 2H 5]-phenylalanine and [ 2H 3]-leucine for the measurement of FSR in exercise-based studies of human skeletal muscle.