Stomata control CO2 uptake for photosynthesis and water loss through transpiration, thus playing a key role in leaf thermoregulation, water-use efficiency (iWUE) and plant productivity. In this work, we investigated the relationship between several leaf traits and hypothesized that stomatal behavior to fast (i.e. minutes) environmental changes co-determines along with steady-state traits the physiological response of grapevine to the surrounding fluctuating environment over the growing season. No relationship between iWUE, heat stress (HS) tolerance and stomatal traits was observed in field grown grapevine, suggesting that other physiological mechanisms are involved in determining leaf evaporative cooling capacity and the seasonal ratio of CO2 uptake (A) to stomatal conductance (gs). Indeed, cultivars that in the field had an unexpected combination of high iWUE but low sensitivity to thermal stress, displayed a quick stomatal closure to light, but a sluggish closure to increased vapor pressure deficit (VPD) levels. This strategy aiming both at conserving water under a high-to-low light transition and in prioritizing evaporative cooling under a low-to-high VPD transition, was mainly observed in Regina and Syrah. Moreover, cultivars with different known responses to soil moisture deficit or high air VPD (isohydric vs anisohydric) had opposite behavior under fluctuating environments, with the isohydric cultivar showing slow stomatal closure to reduced light intensity but quick temporal responses to VPD manipulation. We propose that stomatal behavior to fast environmental fluctuations can play a critical role on leaf thermoregulation and water conservation under natural field conditions in grapevine.