Indirect calorimetry (InCa) estimates whole-body energy expenditure and total substrate oxidation based on O2 consumption and CO2 production, but does not allow for the quantification of oxidation of exogenous substrates with time. To achieve this, we incorporated 13CO2 and 12CO2 gas sensors into a commercial InCa system and aimed to demonstrate their performance and added value. As a performance indicator, we showed the discriminative oscillations in 13CO2 enrichment associated with food intake in mice fed diets containing naturally low (wheat) vs high (maize) 13C enrichment. To demonstrate the physiological value, we quantified exogenous vs total carbohydrate and fat oxidation continuously, in real time in mice varying in fat mass. Diet-induced obese mice were fed a single liquid mixed meal containing 13C-isotopic tracers of glucose or palmitate. Over 13 h, ~70% glucose and ~48% palmitate ingested were oxidised. Exogenous palmitate oxidation depended on body fat mass, which was not the case for exogenous glucose oxidation. We conclude that extending an InCa system with 13CO2 and 12CO2 sensors provides an accessible and powerful technique for real-time continuous quantification of exogenous and whole-body substrate oxidation in mouse models of human metabolic physiology.