Pot-based phenotyping of drought response sometimes maintains suboptimal soil water content by applying high-frequency deficit irrigation (HFDI). We examined the effect of this treatment on water and abscisic acid (ABA) relations of two species (Helianthus annuus and Populus nigra). Suboptimal soil water content was maintained by frequent irrigation, and compared with the effects of withholding water and with adequate irrigation. At the same average whole-pot soil moisture, frequent irrigation resulted in larger soil water content gradients, lower root and xylem ABA concentrations ([X-ABA]), along with higher transpiration rates or stomatal conductance, compared with plants from which water was withheld. [X-ABA] was not uniquely related to transpiration rate or stomatal conductance, as frequently irrigated plants showed partial stomatal closure compared with well-watered controls, without differing in [X-ABA] and, in H. annuus, [ABA] leaf. In two P. nigra genotypes differing in leaf area, the ratio between leaf area and root weight in the upper soil layer influenced the soil water content of this layer. Maintaining suboptimal soil water content alters water relations, which might become dependent on root distribution and leaf area, which influences soil water content gradients. Thus genotypic variation in 'drought tolerance' derived from phenotyping platforms must be carefully interpreted.