Serum iron and serum hepcidin before and after a small iron challenge. Serum samples were obtained from 3 subjects (A-C) over 48 hours, and an iron sulfate dose was given at 8 am of the second day as indicated by the vertical line in all panels. Subject A responded to 65 mg of iron by increasing both serum iron and hepcidin. Subject B was tested on 2 separate occasions, first with 65 mg (solid line), then with 130 mg of iron (dashed line), and only responded to the larger dose by increasing serum iron and hepcidin. Subject C was tested on 2 separate occasions with 65 mg of iron (solid and dashed line) and did not increase either serum iron or hepcidin indicating that the iron dose was not well absorbed. The average time dependence of the 5 serial prechallenge hepcidin measurements in all subjects is also shown in panel C as a bold black line. Hepcidin concentration showed diurnal variation, with noon and 8 pm values significantly higher (●) than baseline 8 am values (1-way repeated measures ANOVA, Holm-Sidak method).