Objective: To examine the prevalence of excretion of urinary isoflavonoids in women and determine any relationships with accustomed macronutrient intake. Design: Volunteers in one of two 4-month studies. Study 1 was a randomised crossover study whereby subjects consumed a placebo or isoflavone supplement for 2 months and crossed over. Study 2 was a parallel design in which subjects consumed a placebo for 1 month and an isoflavone supplement for 3 months. Setting: All subjects were free-living, healthy volunteers. Subjects: A total of 25 (study 1, n = 14; study 2, n = 11) premenopausal women were recruited through advertisements. Interventions: Volunteers were supplemented for 2 months (study 1) or 3 months (study 2) with purified isoflavones (86 mg/day) derived from red clover. Urinary isoflavonoids were measured during the placebo and the second month of isoflavone treatment. Macronutrient intakes were determined from weighed food records. Results: During isoflavone supplementation, the concentration of urinary total isoflavonoids increased by 15-fold (P<0.0001), with 5.4-fold variation between individuals. Multiple linear regression analysis showed that 24% of this variation could be explained by an interaction between dietary fibre and protein (P = 0.047), with a highly significant inverse association between total isoflavonoid concentration and the protein to fibre ratio (r = -0.51, P = 0.009). Conclusions: Supplementation with purified isoflavones results in an increase in urinary isoflavonoid excretion and part of the individual variation in response is associated with an interaction between intakes of protein and dietary fibre. Whether manipulation of these macronutrients could enhance efficacy of isoflavone supplements remains to be determined.