Understanding environmental and physiological controls of the variations in δ13C of CO2 respired (δ13CR) from different compartments of an ecosystem is important for separation of CO2 fluxes and to assess coupling between assimilation and respiration. In a wheat field, over 3 days we characterised the temporal dynamics of δ13CR from shoots and roots, from the soil and from the whole agroecosystem. To evaluate the basis of potential variations in δ13CR, we also measured δ13C in different organic matter pools, as well as meteorological and gas exchange parameters. We observed strong diel variations up to ca. 6% in shoot, root and soil δ13CR, but not in δ13C of the putative organic substrates for respiration, which varied by not more than ca. 1% within 24 h. Whole ecosystem-respired CO2 was least depleted in 13C in the afternoon and most negative in the early morning. We assume that temporally variable respiratory carbon isotope fractionation and changes in fluxes through metabolic pathways, rather than photosynthetic carbon isotope fractionation, governs the δ13C of respired CO2 at the diel scale, and thus provides insights into the metabolic processes related to respiration under field conditions.