Serum iron levels vary throughout the day. Morning levels are generally assumed to be higher than afternoon or evening levels. We studied whether our practice of restricting serum iron collections to the morning was necessary. Serum iron, iron-binding capacity, transferrin saturation, and ferritin levels were determined on blood specimens obtained from 20 healthy adult volunteers at 8 AM, noon, and 4 PM (day 1) and 8 AM (day 2). Although statistically significant differences among mean values for the collection times were observed for iron, iron-binding capacity, and (log) ferritin, no consistent diurnal variation was seen. Morning iron levels were higher than afternoon levels for only half of the subjects. Between-day variation for all 4 analytes was similar to within-day variation. We conclude that the practice of restricting iron specimen collections to a specific time of day does not improve the reliability of the test result.