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The aim of this study was to test the effects of two disparate isonitrogenous, isocaloric pre-exercise feeds on deuterium-oxide (D₂O) derived measures of myofibrillar protein synthesis (myoPS) in humans. <i>Methods:</i> In a double-blind parallel group design, 22 resistance-trained men aged 18 to 35 years ingested a meal (6 kcal&#183;kg^&#8722;1, 0.8 g&#183;kg^&#8722;1 carbohydrate, 0.2 g&#183;kg^&#8722;1 fat) with 0.33 g&#183;kg^&#8722;1 nonessential amino acids blend (NEAA) or whey protein (WHEY), prior to resistance exercise (70% 1RM back-squats, 10 reps per set to failure, 25% duty cycle). Biopsies of <i>M. vastus lateralis</i> were obtained pre-ingestion (PRE) and +3 h post-exercise (POST). The myofibrillar fractional synthetic rate (myoFSR) was calculated via deuterium labelling of myofibrillar-bound alanine, measured by gas chromatography&#8722;pyrolysis&#8722;isotope ratio mass spectrometry (GC-Pyr-IRMS). Data are a mean percentage change (95% CI). <i>Results:</i> There was no discernable change in myoFSR following NEAA (10(&#8722;5, 25) %, <i>p</i> = 0.235), whereas an increase in myoFSR was observed after WHEY (28 (13, 43) %, <i>p</i> = 0.003). <i>Conclusions:</i> Measured by a D₂O tracer technique, a disparate myoPS response was observed between NEAA and WHEY. Pre-exercise ingestion of whey protein increased post-exercise myoPS, whereas a NEAA blend did not, supporting the use of NEAA as a viable isonitrogenous negative control.