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Rationale It was recently shown that the presence of CO2 affects the stable isotope (δ2H and δ18O values) analysis of water vapor via Wavelength‐Scanned Cavity Ring‐Down Spectroscopy. Here, we test how much CO2 is emitted from soil samples and if the CO2 in the headspace influences the isotope analysis with the direct equilibration method by Off‐Axis Integrated Cavity Output Spectroscopy (OA‐ICOS). Methods The headspace above different amounts of sparkling water was sampled, and its stable isotopic composition (δ2H and δ18O values) and CO2 concentration were measured by direct equilibration and by gas chromatography, respectively. In addition, the headspace above soil samples was analyzed in the same way. Furthermore, the gravimetric water content and the loss on ignition were measured for the soil samples. Results The experiment with the sparkling water showed that CO2 does not influence the stable isotope analysis by OA‐ICOS. CO2 was emitted from the soil samples and correlated with the isotopic fractionation signal, but no causal relationship between the two was determined. Instead, the fractionation signal in pore water isotopes can be explained by soil evaporation and the CO2 can be related to soil moisture and organic matter which both enhance microbial activity. Conclusions We found, despite the high CO2 emissions from soil samples, no need for a post‐correction of the pore water stable isotope analysis results, since there is no relation between CO2 concentrations and the stable isotope results of vapor samples obtained with OA‐ICOS. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.