In a constant infusion study of a mass isotope of leucine, two alternative equations are commonly available to calculate amino acid oxidation rate and, thence, whole-body protein synthesis. One, developed by Matthews et al (Am J Physiol Endocrinol Metab. 1980;238:E473-E479), is shown here to require assuming a tracee steady state (TSS), namely, that tracee (unlabeled) amino acid concentrations and fluxes (rates of oxidation and incorporation into protein) are unaltered compared with the preinfusion state. The other, developed by Garlick and coworkers (Melville et al, Metabolism 1989;38:248-255), stems from a protein steady state (PSS) assumption, namely, that protein synthesis is unaffected by the tracer infusion. We derive here a simple expression for the relative difference in whole-body protein synthesis computed from the two assumptions, and a simple test of the validity of TSS in the form of an equality that must be satisfied by plasma measurements at all times. We also propose two experiments to discriminate between the two assumptions. Theoretical reasons and experimental evidence from the literature are offered to support PSS. The two assumptions result in different expressions for fractional synthetic rates (FSRs) of individual or organ proteins-TSS requires the use of tracer-to-tracee ratios and PSS the use of enrichments. An expression is derived here for the relative difference in FSR with TSS vs PSS. For both whole-body synthesis and for FSR, the TSS assumption consistently results in an underestimate, the relative bias roughly equal to the precursor amino acid enrichment.