The isotopic composition of xylem water (δ_X) is of considerable interest for plant source water studies. In-situ monitored isotopic composition of transpired water (δ_T) could provide a nondestructive proxy for δ_X-values. Using flow-through leaf chambers, we monitored 2-hourly δ_T-dynamics in two tropical plant species, one canopy-forming tree and one understory herbaceous species. In an enclosed rainforest (Biosphere 2), we observed δ_T-dynamics in response to an experimental severe drought, followed by a ²H deep-water pulse applied belowground before starting regular rain. We also sampled branches to obtain δ_X-values from cryogenic vacuum extraction (CVE). Daily flux-weighted δ¹⁸O_T-values were a good proxy for δ¹⁸O_X-values under well-watered and drought conditions that matched the rainforest's water source. Transpiration-derived δ¹⁸O_X-values were mostly lower than CVE-derived values. Transpiration-derived δ²H_X-values were relatively high compared to source water and consistently higher than CVE-derived values during drought. Tracing the ²H deep-water pulse in real-time showed distinct water uptake and transport responses: a fast and strong contribution of deep water to canopy tree transpiration contrasting with a slow and limited contribution to understory species transpiration. Thus, the in-situ transpiration method is a promising tool to capture rapid dynamics in plant water uptake and use by both woody and nonwoody species.