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Predicting vegetation responses to global warming and reconstructing them from stable isotope records requires a clear understanding of how they respond to temperature. We investigated leaf carbon-dynamics (gas exchange, non-structural carbohydrate (NSC)) and the hydrogen (δ²H) and oxygen (δ¹⁸O) isotopic composition of leaf water and sugars in well-watered C₃ trees, forbs, grasses, and one C₄ grass across air temperatures from 10 °C to 40 °C at a low VPD. In C₃ species, temperatures ≥30 °C reduced A net , increased R dark , and shifted NSC composition from starch to sugars. Concurrently, apparent δ²H and δ¹⁸O fractionation between leaf water and sugars decreased. δ²H and δ¹⁸O in C₃ plants can be modeled from leaf water isotope composition when accounting for temperature-driven changes in gas exchange and NSC dynamics. These findings reveal heat-induced shifts in carbohydrate metabolism leave distinct isotope signatures, providing a mechanistic basis for improved isotope models to study current and past tree carbon dynamics.