AbstractThe doubly labelled water (DLW) method is widely used to determine energy expenditure. In this work, we demonstrate the addition of the third stable isotope, ¹⁷O, to turn it into triply labelled water (TLW), using the three isotopes measurement of optical spectrometry. We performed TLW (²H, ¹⁸O and¹⁷O) measurements for the analysis of the CO₂ production (r_CO2) of mice on different diets for the first time. Triply highly enriched water was injected into mice, and the isotope enrichments of the distilled blood samples of one initial and two finals were measured by an off-axis integrated cavity output spectroscopy instrument. We evaluated the impact of different calculation protocols and the values of evaporative water loss fraction. We found that the dilution space and turnover rates of ¹⁷O and ¹⁸O were equal for the same mice group, and that values of r_CO2 calculated based on ¹⁸O–²H, or on ¹⁷O–²H agreed very well. This increases the reliability and redundancy of the measurements and it lowers the uncertainty in the calculated r_CO2 to 3% when taking the average of two DLW methods. However, the TLW method overestimated the r_CO2 compared to the indirect calorimetry measurements that we also performed, much more for the mice on a high-fat diet than for low-fat. We hypothesize an extra loss or exchange mechanism with a high fractionation for ²H to explain this difference.