The oxygen isotope signature of water is a powerful tracer of water movement from plants to the global scale. However, little is known about the short-term variability of oxygen iso- topes leaving the ecosystem via transpiration, as high-frequency measurements are lacking. A laser spectrometer was coupled to a gas-exchange chamber directly estimating branch- level fluxes in order to evaluate the short-term variability of the isotopic composition of transpiration (dE) and to investigate the role of isotopic non-steady-state transpiration under natural conditions in cork-oak trees (Quercus suber) during distinct Mediterranean seasons. The measured d18O of transpiration (dE) deviated from isotopic steady state throughout most of the day even when leaf water at the evaporating sites was near isotopic steady state. High agreement was found between estimated and modeled dE values assuming non-steady- state enrichment of leaf water. Isoforcing, that is, the influence of the transpirational d18O flux on atmospheric values, deviated from steady-state calculations but daily means were similar between steady state and non-steady state. However, strong daytime isoforcing on the atmosphere implies that short-term variations in dE are likely to have consequences for large-scale applications, for example, partitioning of ecosystem fluxes or satellite-based applications.