Leaves maintain a pool of non-structural carbohydrates (NSC) whose size can vary over hourly and longer timescales. We tested two long-standing hypotheses regarding potential physiological roles of changes in foliar NSC levels. The first is that soluble NSC play a critical role in osmotic adjustment, with their increase enabling stomatal opening despite daily and seasonal reductions in leaf water potential (Ψ _leaf). The second is that increases in NSC is a sign of excess assimilation relative to sink demand and serves as a signal to downregulate gas exchange. To explore these questions, we monitored the diurnal and seasonal dynamics of gas exchange, Ψ _leaf, osmotic potential, and NSC of irrigated and dehydrated grapevines ( Vitis vinifera) through two consecutive growing seasons. We found that the daily accumulation of soluble sugars constitutes approximately 50% of the daily osmotic adjustment (0.2 MPa), enabling the vines to maintain turgor under low Ψ _leaf. At the same time, the importance of NSC as osmolytes decreased as the season progressed, and they did not contribute to osmotic adjustment when water was withheld. Additionally, there was no negative correlation between NSC and gas exchange, implying that NSC are not the signal for photosynthetic feedback inhibition.