During daylight hours, the isotope composition of leaf water generally approximates steady-state leaf water isotope enrich- ment model predictions. However, until very recently there was little direct confirmation that isotopic steady-state (ISS) transpiration in fact exists. Using isotope ratio infrared spectros- copy (IRIS) and leaf gas exchange systems we evaluated the isotope composition of transpiration and the rate of change in leaf water isotopologue storage (isostorage) when leaves were exposed to variable environments. In doing so, we developed a method for controlling the absolute humidity entering the gas exchange cuvette for a wide range of concentrations without changing the isotope composition of water vapour. The meas- urement system allowed estimation of 18O enrichment both at the evaporation site and for bulk leaf water, in the steady state and the non-steady state. We show that non–steady-state effects dominate the transpiration isoflux even when leaves are at physiological steady state. Our results suggest that a variable environment likely prevents ISS transpiration from being achieved and that this effect may be exacerbated by lengthy leaf water turnover times due to high leaf water contents.