Understanding the influence of vegetation on water storage and flux in the upper soil is crucial in assessing the consequences of climate and land use change. We sampled the upper 20 cm of podzolic soils at 5 cm intervals in four sites differing in their vegetation (Scots Pine (Pinus sylvestris) and heather (Calluna sp. and Erica Sp)) and aspect. The sites were located within the Bruntland Burn long-term experimental catchment in the Scottish Highlands, a low energy, wet environment. Sampling took place on 11 occasions between September 2015 and September 2016 to capture seasonal variability in isotope dynamics. The pore waters of soil samples were analyzed for their isotopic composition (δ2H and δ18O) with the direct-equilibration method. Our results show that the soil waters in the top soil are, despite the low potential evaporation rates in such northern latitudes, kinetically fractionated compared to the precipitation input throughout the year. This fractionation signal decreases within the upper 15 cm resulting in the top 5 cm being isotopically differentiated to the soil at 15–20 cm soil depth. There are significant differences in the fractionation signal between soils beneath heather and soils beneath Scots pine, with the latter being more pronounced. But again, this difference diminishes within the upper 15 cm of soil. The enrichment in heavy isotopes in the topsoil follows a seasonal hysteresis pattern, indicating a lag time between the fractionation signal in the soil and the increase/decrease of soil evaporation in spring/autumn. Based on the kinetic enrichment of the soil water isotopes, we estimated the soil evaporation losses to be about 5 and 10 % of the infiltrating water for soils beneath heather and Scots pine, respectively. The high sampling frequency in time (monthly) and depth (5 cm intervals) revealed high temporal and spatial variability of the isotopic composition of soil waters, which can be critical, when using stable isotopes as tracers to assess plant water uptake patterns within the critical zone or applying them to calibrate tracer-aided hydrological models either at the plot to the catchment scale.