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To validate 24 h dietary recall of fruit intake by measuring the total 24 h excretion of 10 different flavonoids in 24 h urine during an intervention with free fruit at workplaces.Employees at workplaces offering a free-fruit program, consisting of daily free and easy access to fresh fruit, and controls employees at workplaces with no free-fruit program were enrolled in this validation study (n=103). Dietary intake was assessed by using a 24 h dietary recall questionnaire at baseline and approximately 5 months later. Ten flavonoids, quercetin, isorhamnetin, tamarixetin, kaempferol, hesperetin, naringenin, eriodictyol, daidzein, genistein, and phloretin, were measured using HPLC-electrospray ionization-MS.The 24 h urinary excretion of total flavonoids and the estimated intake of fruits were significantly correlated (rs=0.31, P<0.01). The dietary intake of citrus fruits and citrus juices was significantly correlated with total excretion of citrus specific flavonoids (rs=0.28, P<0.01), and orange was positively correlated with naringenin (rs=0.24, P<0.01) and hesperetin (rs=0.24, P<0.01). Phloretin in urine was correlated with apple intake (rs=0.22, P<0.01) and also with overall estimated intake of fruit (rs=0.22, P<0.01).This study shows that a 24 h dietary recall can be used as a valid estimate of the intake of fruits in agreement with an objective biomarker of fruit intake in free fruit at workplace interventions.