Recent studies have demonstrated that the oral 13C bicarbonate technique (o13CBT) can be used as a minimally invasive method for estimation of CO2 production (RCO2) and energy expenditure (EE) in dogs. However, correct estimates for the 13C recovery factor (RF) and the respiratory quotient (RQ) are of vital importance for achieving accurate estimates of RCO2 and EE. The objective of this study was to validate the o13CBT against indirect calorimetry (IC) under different conditions in dogs. The study included three experiments in which the effect of different feeding time relative to measurements, different dietary composition and different ambient temperature, on the estimates for the RF and RQ, were investigated. The RF and RQ values were not significantly affected by the different conditions in the three experiments. However, the variation in all experiments was large. Standardizing the measurements as much as possible is necessary and therefore, measurements after fasting over the night are suggested.