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The measurement of soil CO 2 fluxes and their isotopic composition is increasingly used to study and distinguish respiration sources in the soil profile. The influence of non-steady state processes on soil respiratory δ 13 CO 2 values, such as diffusive fractionation, remains unclear but is suggested to be strong, especially in arid ecosystems. In this study, an in situ Fourier transform infrared spectrometer was coupled to automated soil flux chambers and installed in an arid grassland in Italy. Hourly flux and concentration measurements of CO 2 and its isotopic composition were taken over bare soil and senescent grass material. CO 2 fluxes were stable at night (∼1 μmol m −2 s −1 ), and showed peak emissions during the day (up to 10 μmol m −2 s −1 ). Respiratory δ 13 CO 2 values showed a diel pattern. Daytime respiratory δ 13 CO 2 values were on average −24.8‰ and nighttime values were on average −26.9‰, but showed significantly more depleted values during nights with strong increase in atmospheric CO 2 . We hypothesize that the observed diel variation in respiratory δ 13 CO 2 values was a consequence of the non-steady state process “diffusive fractionation” induced by the increasing atmospheric CO 2 concentrations at night. [ABSTRACT FROM AUTHOR]