Summary Recent studies showed that the urinary excretion of the water-soluble vitamins can be useful as a nutritional index. To determine how fasting affects urinary excretion of water-soluble vitamins, a human study and an animal experiment were conducted. In the human study, the 24-h urinary excretion of water-soluble vitamins in 12 healthy Japanese adults fasting for a day was measured. One-day fasting drastically decreased urinary thiamin content to 30%, and increased urinary riboflavin content by 3-fold. Other water-soluble vitamin contents did not show significant change by fasting. To further investigate the alterations of water-soluble vitamin status by starvation, rats were starved for 3 d, and water-soluble vitamin contents in the liver, blood and urine were measured during starvation. Urinary excretion of thiamin, riboflavin, vitamin B 6 metabolite 4-pyridoxic acid, nicotinamide metabolites and folate decreased during starvation, but that of vitamin B 12 , pantothenic acid and biotin did not. As for blood vitamin levels, only blood vitamin B 1 , plasma PLP and plasma folate levels decreased with starvation. All water-soluble vitamin contents in the liver decreased during starvation, whereas vitamin concentrations in the liver did not decrease. Starvation decreased only concentrations of vitamin B 12 and folate in the skeletal muscle. These results suggest that water-soluble vitamins were released from the liver, and supplied to the peripheral tissues to maintain vitamin nutrition. Our human study also suggested that the effect of fasting should be taken into consideration for subjects showing low urinary thiamin and high urinary riboflavin. Key Words human, rats, fasting, urine, biomarker A biological assay technique to assess water-soluble vitamins in healthy human subjects was reported by Melnick et al . in 1945 ( 1 ), and this technique became popular in the 1960 s and 1970 s. Urinary excretion of thiamin, riboflavin and niacin metabolites has been used for setting Dietary Reference Intakes (DRIs) of vitamin B 1 , vitamin B 2 and niacin in the USA and Japan ( 2 , 3 ). Recent validation studies have developed the urinary compounds as nutritional markers to estimate nutrient intakes. We have recently reported that each of the water-soluble vitamins or its metabolite levels in the 24-h urine are strongly correlated with its intake when young women consume the standard Japanese diet with several amounts of water-soluble vitamin ( 4 ). Thirtyday mean thiamin levels in 24-h urine are also highly correlated with mean intake of vitamin B 1 ( 5 ). These findings show that urinary excretion of water-soluble vitamins reflects their intakes when subjects constantly take certain amounts of water-soluble vitamins. It is well known that pharmacological doses of water-soluble vitamins acutely increase their excretion to urine, and that a chronic water-soluble vitamin restricted diet reduces its levels in urine and blood ( 6 ). Effects of acute deprivation of water-soluble vitamins on their urinary excretion have a...