Recent insights into fractionation during dark respiration and rapid dynamics in isotope signatures of leaf- and ecosystem-respired CO2 indicate the need for new methods for high time-resolved measurements of the isotopic signature of respired CO2 (δ13Cres). We present a rapid and simple method to analyse δ13Cres using an in-tube incubation technique and an autosampler for small septum-capped vials. The effect of storage on the δ18O and δ13C ratios of ambient CO2 concentrations was tested with different humidity and temperatures. δ13C ratios remained stable over 72 h, whereas δ18O ratios decreased after 24 h. Storage at 4°C improved the storage time for δ18O. Leaves or leaf discs were incubated in the vials, flushed with CO2-free air and respired CO2 was automatically sampled within 5 min on a µGas autosampler interfaced to a GV-Isoprime isotope ratio mass spectrometer. Results were validated by simultaneous on-line gas-exchange measurements of δ13Cres of attached leaves. This method was used to evaluate the short-term (5–60 min) and diurnal dynamics of δ13Cres in an evergreen oak (Quercus ilex) and a herb (Tolpisbarbata). An immediate depletion of 2–4‰ from the initial δ13Cres value occurred during the first 30 min of darkening. Q. ilex exhibited further a substantial diurnal enrichment in δ13Cres of 8‰, followed by a progressive depletion during the night. In contrast, T. barbata did not exhibit a distinct diurnal pattern. This is in accordance with recent theory on fractionation in metabolic pathways and may be related to the different utilisation of the respiratory substrate in the fast-growing herb and the evergreen oak. These data indicate substantial and rapid dynamics (within minutes to hours) in δ13Cres, which differed between species and probably the growth status of the plant. The in-tube incubation method enables both high time-resolved analysis and extensive sampling across different organs, species and functional types. Copyright © 2007 John Wiley & Sons, Ltd.