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Background Red clover is an important source of isoflavones; which has been made commercially available as dietary supplements for the treatment of menopausal symptoms. Bioavailability and metabolism of these red clover isoflavones (RCI) have not been studied in detail. Fructooligosaccharides (FOS) stimulate the growth of intestinal bacteria and play an important role in the formation of certain isoflavone metabolites, such as equol and O -desmethylangolensin. Objective To determine the bioavailability of RCI metabolites and analyse whether FOS supplementation could influence their bioavailability. Methods Seventeen healthy adults were enrolled in the study carried out in two periods. In the first, compound bioavailability was determined after consumption of 80 mg of RCI (MF11RCE). In the second, a 6-day supplementation of 2 × 3000 mg/day of FOS was administered before isoflavone consumption. Results Biochanin A and formononetin were rapidly absorbed and both reached maximum concentrations at an average of 5–7 h. Demethylation was a major reaction in the metabolic pathway. Daidzein serum level peaked after about 12.6 h. Supplementation with FOS led to a significant decrease in the bioavailability of daidzein, dihydroformononetin, dihydrogenistein and dihydrodaidzein. An increase in equol production was also observed which did not reach statistical significance ( p > 0.05). Conclusion This study is the first to provide detailed data on RCI bioavailability in humans and determine no influence of FOS yet a trend toward increased equol production. More research is warranted involving a greater sample size. [ABSTRACT FROM AUTHOR]