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Human biotin requirements are unknown and the identification of reliable markers of biotin status is necessary to fill this knowledge gap. Here we used an outpatient feeding protocol to create states of biotin deficiency, sufficiency, and supplementation in 16 healthy men and women. Twenty possible markers of biotin status were assessed including the abundance of biotinylated carboxylases in lymphocytes, the expression of genes from biotin metabolism, and the urinary excretion of biotin and organic acids. Only the abundance of biotinylated 3-methylcrotonyl-CoA carboxylase (holo-MCC) and propionyl-CoA carboxylase (holo-PCC) allowed for distinguishing among all three levels of biotin intake. The urinary excretion of biotin reliably identified biotin-supplemented subjects, but did not distinguish between biotin-depleted and biotin-sufficient individuals. The urinary excretion of 3-hydroxyisovaleric acid (3-HIA) detected some biotin-deficient subjects, but produced a meaningful number of false negative results and did not distinguish between biotin-sufficient and biotin-supplemented individuals. The urinary excretion of 3-HIA might be a more reliable marker of biotin status, if used in combination with urinary citrate (positively associated with biotin intake) and malate (negatively associated with biotin intake). None of the other organic acids that were tested were useful markers of biotin status. Likewise, the abundance of mRNA coding for biotin transporters, holocarboxylase synthetase, and biotin-dependent carboxylases in lymphocytes were not different among treatment groups. Generally, data sets were characterized by variations that exceeded those seen in studies in cell cultures. We conclude that holo-MCC and holo-PCC are the most reliable, single markers of biotin status tested in this study.