Changes in leaf carbon and nitrogen isotope composition (δ ¹³ C and δ ¹⁵ N values) and the accumulation of epicuticular lipids have been associated with plant responses to water stress. We investigated their potential use as indicators of early plant water deficit in two grapevine (Vitis vinifera L.) cultivars, Chasselas and Pinot noir, that were field-grown under well-watered and water-deficient conditions. We tested the hypothesis that the bulk δ ¹³ C and δ ¹⁵ N values and the concentrations of epicuticular fatty acids may change in leaves of similar age with the soil water availability. For this purpose, leaves were sampled at the same position in the canopy at different times (phenological stages) during the 2014 growing season. Bulk dry matter of young leaves from flowering to veraison had higher δ ¹³ C values, higher total nitrogen content, and lower δ ¹⁵ N values than old leaves. In both cultivars, δ ¹⁵ N values were strongly correlated with plant water deficiency, demonstrating their integration of the plant water stress response. δ ¹³ C values recorded the water deficiency only in those plants that had not received foliar organic fertilization. The soil water deficiency triggered the accumulation of C _>26 fatty acids in the cuticular waxes. The compound-specific isotope analysis (CSIA) of fatty acids from old leaves showed an increase in δ ¹³ C among the C ₁₆ -C ₂₂ chains, including stress signaling linoleic and linolenic acids. Our results provide evidence for leaf ¹³ C-enrichment, ¹⁵ N-depletion, and enhanced FA-chain elongation and epicuticular accumulation in the grapevine response to water stress. The leaf δ ¹³ C and δ ¹⁵ N values, and the concentration of epicuticular fatty acids can be used as reliable and sensitive indicators of plant water deficit even when the level of water stress is low to moderate. They could also be used, particularly the more cost-efficient δ ¹³ C and δ ¹⁵ N measurements, for periodic biogeochemical mapping of the plant water availability at the vineyard and regional scale.