Monitoring early tree physiological responses to drought is key to understanding progressive impacts of drought on forests and identifying resilient species. We combined drone-based multispectral remote sensing with measurements of tree physiology and environmental parameters over two growing seasons in a 100-y-old Pinus sylvestris forest subject to 17-y of precipitation manipulation. Our goal was to determine if drone-based photochemical reflectance index (PRI) captures tree drought stress responses and whether responses are affected by long-term acclimation. PRI detects changes in xanthophyll cycle pigment dynamics, which reflect increases in photoprotective non-photochemical quenching activity resulting from drought-induced photosynthesis downregulation. Here, PRI of never-irrigated trees was up to 10 times lower (higher stress) than PRI of irrigated trees. Long-term acclimation to experimental treatment, however, influenced the seasonal relationship between PRI and soil water availability. PRI also captured diurnal decreases in photochemical efficiency, driven by vapour pressure deficit. Interestingly, 5 years after irrigation was stopped for a subset of the irrigated trees, a positive legacy effect persisted, with lower stress responses (higher PRI) compared with never-irrigated trees. This study demonstrates the ability of remotely sensed PRI to scale tree physiological responses to an entire forest and the importance of long-term acclimation in determining current drought stress responses.