Olive trees have a unique reproductive pattern marked by biennial fruiting. This study delves into the repercussions of alternate fruit bearing on the water relations of olive trees and the associated ecophysiological mechanisms. The experiment spanned two consecutive years: the "ON" year, characterized by a high crop load, and the "OFF" year, marked by minimal fruit production. Key ecophysiological parameters, including sap flow, stomatal conductance, and photosynthetic rate, were monitored in both years. Pre-dawn water potential was measured using continuous stem psychrometers and the pressure chamber technique. Biochemically, non-structural carbohydrate concentrations (starch, sucrose, and mannitol) and the carbon-stable isotope ratio (δ13C) in olive leaves were examined. Results unveiled heightened leaf gas exchange during the "ON" year, leading to increased water consumption and photosynthetic rates. This escalated water usage during the "ON" year significantly reduced leaf water potential. Sucrose and starch concentrations were notably elevated in the "ON" year, with no significant impact on mannitol concentration. Regarding the carbon stable isotope ratio, leaves from the "OFF" year exhibited significantly higher δ13C abundance, indicative of heightened resistance to the CO2 pathway from the atmosphere to carboxylation sites compared to the "ON" year plants.