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Significant water loss occurs throughout the night via partially open stomata in many C₃ and C₄ plant species. Although apparently wasteful in terms of water use, nighttime transpiration (Enight) is hypothesized to benefit plants by enhancing nutrient supply. We tested the hypothesis that plants with greater Enight would have improved plant nutrient status and greater fitness, estimated as pre-bolting biomass, for Arabidopsis thaliana. Two very different levels of Enight were generated in plants by exposing them to high vs low nighttime leaf-to-air vapor pressure deficits (VPDleaf) in controlled environment chambers. An assessment of responses of nighttime leaf conductance (gnight) to VPDleaf indicated that Enight differed by at least 80% between the treatments. This large difference in Enight, imposed over the entire vegetative growth phase of Arabidopsis, had no effect on leaf nutrient content (N, Ca, K) or pre-bolting rosette biomass. The lack of response to differences in Enight held true for both a high and a low nitrogen (N) treatment, even though the low N treatment decreased leaf N and biomass by 40-60%. The N treatment had no effect on gnight. Thus, higher Enight did not provide a nutrient or growth benefit to Arabidopsis, even when the plants were N-limited.