The stable carbon isotope ratio of exhaled CO(2) (delta(13)C(breath)) reflects the isotopic signature of the combusted substrate and is, therefore, suitable for the non-invasive collection of dietary information from free-ranging animals. However, delta(13)C(breath) is sensitive to changes in ingested food items and the mixed combustion of exogenous and endogenous substrates. Therefore, experiments under controlled conditions are pivotal for the correct interpretation of delta(13)C(breath) of free-ranging animals. We measured delta(13)C(breath) in fasted and recently fed insectivorous Myotis myotis (Chiroptera) to assess the residence time of carbon isotopes in the pool of metabolized substrate, and whether delta(13)C(breath) in satiated individuals levels off at values similar to the dietary isotope signature (delta(13)C(diet)) in insect-feeding mammals. Mean delta(13)C(breath) of fasted individuals was depleted by -5.8 per thousand (N=6) in relation to delta(13)C(diet). After feeding on insects, bats exchanged 50% of carbon atoms in the pool of metabolized substrates within 21.6+/-10.5 min, which was slower than bats ingesting simple carbohydrates. After 2 h, delta(13)C(breath) of satiated bats levelled off at -2.6 per thousand below delta(13)C(diet), suggesting that bats combusted both exogenous and endogenous substrate at this time. A literature survey revealed that small birds and mammals metabolize complex macronutrients at slower rates than simple macronutrients. On average, delta(13)C(breath) of fasting birds and mammals was depleted in (13)C by -3.2+/-2.0 per thousand in relation to delta(13)C(diet). delta(13)C(breath) of satiated animals differed by -0.6+/-2.3 per thousand from delta(13)C(diet) when endogenous substrates were not in isotopic equilibrium with exogenous substrates and by +0.5+/-1.8 per thousand (N=6 species) after endogenous substrates were in isotopic equilibrium with exogenous substrates.