Hydrogen and oxygen stable isotope ratios in plant water are crucial for tracing water sources in ecohydrology. Traditionally, cryogenic vacuum distillation (CVD) has been favored for extracting plant water for isotopic analysis, though its effect on isotopic values is debated. Recently, the Cavitron technique has been suggested as a superior alternative to CVD for extracting xylem water, based on its performance in comparative studies. However, these studies have not systematically evaluated the impact of varying CVD protocols (temperature and time) against the Cavitron method. In our research, we analyzed the isotopic composition of xylem water from the same tree using varying CVD protocols, with different temperatures and extraction times (60°C and 60, 120, 240, 360 min, 100°C and 30, 60, 120, 240 min, 140°C and 15, 30, 60, 120, 240 min, 200°C and 15, 30 min), and compared these with Cavitron extraction results. Our findings indicate that isotopic values for CVD-extracted xylem water increase with both extraction time and temperature. When extraction efficiency reached over 98%, there was no significant isotopic difference among the CVD protocols. However, at lower temperatures, CVD-extracted xylem water showed more negative δ2H and lc-excess values compared to higher temperatures. Cavitron extraction yielded results similar to CVD at lower temperatures and shorter times but was more negative in δ2H and δ18O to CVD at higher temperatures and longer times. These results suggest that reconsidering the replacement of CVD with Cavitron for plant water extraction may be premature without further comprehensive comparisons.