Tree water uptake processes and ecohydrological travel times have gained more attention in recent ecohydrological studies. In situ measurement techniques for stable water isotopes offer great potential to investigate these processes but have not been applied much to tree xylem and soils so far. Here, we used in situ probes for stable water isotope measurements to monitor the isotopic signatures of soil and tree xylem water before and after two deuterium-labeled irrigation experiments. To show the potential of the method, we tested our measurement approach with 20-year-old trees of three different species (Pinus pinea, Alnus incana and Quercus suber). They were planted in large pots with homogeneous soil in order to have semi-controlled experimental conditions. Additional destructive sampling of soil and plant material allowed for a comparison between destructive (cryogenic vacuum extraction and direct water vapor equilibration) and in situ isotope measurements. Furthermore, isotope-tracer-based ecohydrological travel times were compared to travel times derived from sap flow measurements. The time to first arrival of the isotope tracer signals at 15 cm stem hight were ca. 17 h for all tree species and matched well with sap-flow-based travel times. However, at 150 cm stem height tracer-based travel times differed between tree species and ranged between 2.4 and 3.3d. Sap-flow-based travel times at 150cm stem hight were ca. 1.3d longer than tracer-based travel times. The isotope signature of destructive and in situ isotope measurements differed notably, which suggests that the two types of techniques sampled water from different pools. In situ measurements of soil and xylem water were much more consistent between the three tree pots (on average standard deviations were smaller by 8.4‰ for δ2H and by 1.6‰ for δ18O for the in situ measurements) and also among the measurements from the same tree pot in comparison to the destructive methods (on average standard deviations were smaller by 7.8‰ and 1.6‰ for δ2H and δ18O, respectively). Our study demonstrates the potential of semi-controlled large-scale pot experiments and very frequent in situ isotope measurements for monitoring tree water uptake and ecohydrological travel times. It also shows that differences in sampling techniques or sensor types need to be considered when comparing results of different studies and within one study using different methods.