We analysed the stable carbon isotope ratio in exhaled CO2 (δ13Cbreath) of free-ranging vampires to assess the type of metabolized substrate (endogenous or exogenous substrate) and its origin, i.e. whether the carbon atoms came from a C4 food web (grass and cattle) or the C3 food web in which they were captured (a rainforest remnant and its mammals). For an improved understanding of factors influencing the δ13Cbreath of vampires, we conducted feeding experiments with captive animals. The mean δ13Cbreath of starved bats was depleted in 13C in relation to the diet by 4.6‰ (n = 10). Once fed with blood, δ13Cbreath levelled off within a short time approximately 2.2‰ above the stable carbon isotope signature of the diet. The median time required to exchange 50% of the carbon atoms in exhaled CO2 with carbon atoms from the ingested blood was 18.6 min (mean 29.5 ± 19.0 min, n = 5). The average δ13C of wing membrane and fur in free-ranging vampire bats suggested that bats almost exclusively foraged for cattle blood during the past weeks. The δ13Cbreath of the same bats averaged −19.1‰. Given that all free-ranging vampires were starving and that the δ13C of cattle was more in enriched in 13C by 5–6‰ than the δ13Cbreath of vampires, we conclude that the vampire bats of our study metabolised fat that was predominantly built from carbon atoms originating from cattle blood. Since δ13C of wing membrane and fur integrates over weeks and months respectively and δ13Cbreath over hours and days, we also conclude that vampire bats of the studied population consistently ignored rainforest mammals and chose cattle as their prey during and prior to our study.