Increased hydration is recommended as healthy habit with several merits. However, supportive data are sparse. To assess the efficacy of increased daily water intake, we tested the effect of water supplementation on biomarkers in blood, urine, and saliva. Twenty-four healthy Japanese men and 31 healthy Japanese women with fasting blood glucose levels ranging from 90–125 mg/dL were included. An open-label, two-arm, randomized controlled trial was conducted for 12 weeks. Two additional 550 mL bottles of water on top of habitual fluid intake were consumed in the intervention group. The subjects drank one bottle of water (550 mL) within 2 h of waking, and one bottle (550 mL) 2 h before bedtime. Subjects increased mean fluid intake from 1.3 L/day to 2.0 L/day, without changes in total energy intake. Total body water rate increased with associated water supplementation. There were no significant changes in fasting blood glucose and arginine vasopressin levels, but systolic blood pressure was significantly decreased in the intervention group. Furthermore, water supplementation increased body temperature, reduced blood urea nitrogen concentration, and suppressed estimated glomerular filtration rate reduction. Additionally, existence of an intestinal microbiome correlated with decreased systolic blood pressure and increased body temperature. Habitual water supplementation after waking up and before bedtime in healthy subjects with slightly elevated fasting blood glucose levels is not effective in lowering these levels. However, it represents a safe and promising intervention with the potential for lowering blood pressure, increasing body temperature, diluting blood waste materials, and protecting kidney function. Thus, increasing daily water intake could provide several health benefits.