Understanding ecosystem water fluxes has gained increasing attention, as climate scenarios predict a drier environment for many parts of the world. Evaporative enrichment of 18O (Δ18O) of leaf water and subsequent enrichment of plant organic matter can be used to characterize environmental and physiological factors that control evaporation, based on a recently established mechanistic model. In a Pinus sylvestris forest, we measured the dynamics of oxygen isotopic composition (δ18O) every 6 h for 4 d in atmospheric water vapour, xylem sap, leaf water and water-soluble organic matter in current (N) and previous year (N-1) needles, phloem sap, together with leaf gas exchange for pooled N and N-1 needles, and relevant micrometeorological variables. Leaf water δ18O showed strong diel periodicity, while δ18O in atmospheric water vapour and in xylem sap showed little variation. The Δ18O was consistently lower for N than for N-1 needles, possibly related to phenological stage. Modelled leaf water Δ18O showed good agreement with measured values when applying a non-steady state evaporative enrichment model including a Péclet effect. We determined the time lags between δ18O signals from leaf water to water-soluble foliar organic matter and to phloem sap at different locations down the trunk, which clearly demonstrated the relevance of considering these time-lag effects for carbon transport, source-sink and carbon flux partitioning studies.