The isotopic composition of xylem water (δX) is of considerable interest for plant source water studies. In-situ monitored isotopic composition of transpired water (δT) could provide a nondestructive proxy for δX-values. Using flow-through leaf chambers, we monitored 2-hourly δT-dynamics in two tropical plant species, one canopy-forming tree and one understory herbaceous species. In an enclosed rainforest (Biosphere 2), we observed δT-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 δX-values from cryogenic vacuum extraction (CVE). Daily flux-weighted δ18OT-values were a good proxy for δ18OX-values under well-watered and drought conditions that matched the rainforests water source. Transpiration-derived δ18OX-values were mostly lower than CVE-derived values. Transpiration-derived δ2HX-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.