In in vivo metabolic studies, quantifying the flux of a nutrient and/or the carbon atoms through the liver implies the analytical determination of nutrient concentrations before and after crossing the liver and of blood or plasma flux. There is a measurement uncertainty (MU) associated with each input quantity, which may compromise accurate physiological interpretations of the results. The objective of this work was to calculate the expanded MU of net fluxes of nutrients and carbon atoms through the liver of ruminants and to identify the main components that affect MUs. To this end, all quantities (i.e., nutrient concentrations, para-aminohippuric acid concentrations, hematocrit, time of infusion, and others) that influenced the final results were identified and quantified. Their associated MUs were calculated with Type A or Type B approaches. An expanded MU model was developed by combining these two approaches. In general, the relative expanded MU of the net flux of nutrients calculated in plasma was lower than that in blood. For both plasma and blood fluxes, the main components that increased MUs were identified. The net hepatic flux of carbon atoms, for example, was estimated to be (63 +/- A 229) mmol min(-1). This high expanded MU was mainly a result of acetate concentration measurements in hepatic and portal veins. Priority strategies to reduce MUs, such as selecting an adequate matrix or using more accurate measurement methods, were identified.