Fasting serum folate levels are commonly used in assessing folate status and also in estimating the bioavailability of synthetic folic acid and food folate. Previous work has shown that serum folate more than doubles in concentration during a 48 hour fast. Following a 24-hour standardisation procedure, serum and urinary folate levels were measured in nine healthy female volunteers fasting for 36 hours and in the first 6 hours during refeeding. Serum folate concentration increased from a mean of 14.8 ng/ml to 29.3 ng/ml during the 36 hour fast and fell to 22.1 ng/ml during the 6 hour refeeding period. The rise in serum folate concentration during the fast was negatively related to serum folate concentration at 0 hours fasting. It is hypothesised that the enterohepatic recirculation of folate plays an important role in the underlying physiological mechanism. These findings highlight the need to standardise energy intake to control for hepatic influences on folate metabolism in future studies assessing the bioavailability of folate in food.