Short-term triglyceride (TG) synthesis was measured over 48 h in four healthy males from the incorporation rate of deuterium in body water into plasma TG. Subjects drank 0.7 g D2O kg−1 estimated body water (99.8 atom% excess), followed by water containing 1.4 g D2O kg−1 water to maintain plasma deuterium enrichment at plateau. Blood samples (20 ml) were obtained before dosing and every 4 h thereafter. Subjects self-selected three meals each day. TG from each time point were separated from plasma lipids by thin-layer chromatography and combusted to water and CO2. Combustion water was vacuum distilled into Zn-containing Pyrex tubes, reduced to hydrogen gas, and analyzed for deuterium enrichment by isotope ratio mass spectrometry. Deuterium enrichment of TG increased over the 48 h study period for all four subjects studied. Superimposed on this increase were short-term non-periodic fluctuations in enrichment reflecting dietary influx and intra-individual differences in TG metabolism. The TG fractional synthetic rate (FSR) was calculated using linear and mono-exponential models. Triglyceride FSR of the subjects over the first 24 h of the study was 0.0702 ± 0.0048 day−1 (mean ± SEM) by the linear model and 0.0728 ± 0.0051 day−1 by the exponential model. Deuterium enrichment reached a plateau on day 2, indicative of continuing TG synthesis in a saturated body water pool. These results are consistent with the notion of meal-dependent variability in TG synthesis into a small rapid turnover plasma TG pool.