Soils are the largest pool of carbon (C) in terrestrial ecosystems with labile C being particularly vulnerable to loss. In this study we incubated a range of soils in both the short- (minutes) and long-term (months) to assess the loss of labile soil C by measuring the isotopic signature of soil respired CO2 (∂13CO2). Strong temporal trends in ∂13CO2 values were observed following soil disturbance: ∂13CO2 rapidly changed from a range of −22.5 to −23.9‰ to −25.8 to −27.5‰ during short-term incubations and reverted back to the initial values in long-term incubations. The shifts in ∂13CO2 over the course of soil incubations were consistent with changes in labile C substrate utilization following the disturbance of sampling the soil. An independent experimental approach which immobilised labile soil C onto allophane and included chemical extractions as a measure of extractable C in soils also confirmed this interpretation. Collectively, these results indicate that the isotopic analysis of respired CO2 can be a powerful technique which enables us to probe mechanisms and examine the consequences of disturbance on the labile component of soil C.