Adult muscle consists of multinucleated myofibers that originate from myogenic precursor cells during embryonic development. The gene programme of muscle differentiation is well described, but currently there is paucity of data regarding protein synthetic responses during muscle cell fusion. We used stable isotope (deuterium oxide, D2O) labelling of murine C2C12 muscle cells in vitro to investigate the synthesis of individual proteins during early and latter stages of differentiation. Upon reaching 80 % confluency, myoblasts were transferred to differentiation medium supplemented with D2O to label newly synthesised proteins during early (0-24 h) or later (72-96 h) periods of differentiation. Samples were analysed by liquid chromatography-mass spectrometry and data from control and D2O-labelled cells were used to calculate protein fractional synthesis rates (FSR). Samples were also spiked with a yeast protein (alcohol dehydrogenase) to facilitate reporting of synthesis and abundance data in absolute terms. Proteomic analysis of 152 proteins and one-way ANOVA (n=3, per group) detected 55 proteins that exhibited significant (P<0.05, false discovery rate of <1 %) differences between early and late differentiation. Ribosomal proteins were enriched during early differentiation, whereas myofibrillar and metabolic enzymes became more abundant during later differentiation. The median (first – third quartile) FSR (%/h) during early differentiation 4.1 (2.7-5.3) was ~2-fold greater than during later differentiation 1.7 (1.0-2.2), which equates to absolute (fmol/h/ug total protein) synthesis rates of 0.64, (0.38-1.2) and 0.28, (0.1-0.5), respectively. When expressed in relative (%/h) terms the top 5 synthesised proteins during early differentiation were cytosolic actin (ACTC), myc box-dependent-interacting protein 1 (BIN1), myosin light chain 6 (MYL6), protein phosphatase 1-alpha (PP1A) and peroxiredoxin-5 (PRDX5), whereas ubiquitin-40S ribosomal protein S27a (RS27A), stress-induced-phosphoprotein 1 (STIP 1), PP1A, troponin C, slow (TNNC1) and ADP-ribosylation factor 1 (ARF1) were ranked the highest in later differentiation. The interpretation of data was different when expressed in molar (fmol/h/ug total protein) terms. ACTC, alpha enolase (ENOA) and RS27A, were all top-ranked proteins in both early and later differentiation. Additionally, glyceraldehyde 3-phosphate dehydrogenase (G3P; 4.8 fmol/h/ug protein) and Peptidyl-prolyl cis-trans isomerase A (PPIA; 4.0 fmol/h/ug protein) were also high in early, while Desmin (DESM; 1.8 fmol/h/ug protein), and vimentin (VIME; 1.6 fmol/h/ug protein) were high later during differentiation. In conclusion, the biological interpretation of protein synthesis data can differ when results are reported in absolute or relative terms. This has consequences when studying cellular adaptation or the stoichiometry of multi-protein complexes.