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Stem refilling has been studied in many forest species, but its impact on olive trees remains underexplored. This study aims to examine the effect of varying fruit loads on stem refilling rates in olive trees. The experiment was conducted in a commercial olive orchard over two years, characterized by a biennial bearing cycle with minimal fruit production in 2021 (“OFF” year) and maximal fruit production in 2022 (“ON” year). Sap flow sensors measured the water volume differences traversing the apex and base of main branches in two experimental trees. Tree water status was monitored using psychrometers, and soil moisture content was continuously recorded. Results suggest that alternate bearing significantly affects the stem refilling process, while soil moisture availability also plays a pivotal role. During the “ON” year, water reserve consumption increased to 63.6% compared to the “OFF” year to meet the water requirements of developing fruits. Replenishment occurred at various times throughout the 24 h period, including early morning, afternoon, and night when stomatal conductance measurements indicated stomatal closure. During the “ON” year, olive trees experienced intense nocturnal replenishment of reserves, regardless of soil moisture, water vapor pressure deficit, or fruit development stage. These findings offer novel insights into olive trees’ rehydration dynamics that can be used to optimize irrigation scheduling and improve water use efficiency.