Pulse-labelling of plants in a ¹⁴CO₂ or ¹³CO₂ atmosphere is a useful tool for investigating C allocation in plants and soils, revelation of C sources in soil organisms, as well as for CO₂ partitioning studies. Recently, these labelling experiments have been used to produce isotopically labelled biomass of plants or microorganisms for the investigation of C dynamics in these organisms or in soil. However, it remains unknown whether these labelling approaches may affect the composition of plant constituents that react to modifications of environmental conditions during biosynthesis. Lipids as primary biosynthesis products and main components of plant waxes are well known to react fast to environmental changes resulting in a modified lipid composition. In this study, we demonstrate that lipid composition may be only slightly affected by the labelling procedure, especially, when only short pulses (only a few hours) are applied and when the sampling does not occur immediately after the labelling. While the differences of plant lipid compositions are obviously modified not as a result of isotopic pulses, the environmental conditions of plants grown under controlled laboratory conditions have a significant effect leading to a shift of the distribution pattern of plant lipids compared to the lipid composition of plants grown under field conditions.