To investigate whether overnight storage causes significant changes in whole blood, we measured serum and plasma concentrations of vitamins A, C, and E, carotenoids, lipids, and testosterone in whole blood samples stored in a refrigerator at 4 degrees C for 6 and 24 h before centrifugation, aliquoting and freezing them at -70 degrees C or below. In comparison with baseline samples prepared within 2 h, the mean percentage changes at 24 h were: -3.0% to +1.0% for retinol, alpha-tocopherol, and gamma-tocopherol in plasma; -8.7% to -0.1% for carotenoids in plasma; -7.2% for vitamin C in plasma and -1.8% for vitamin C in serum; -2.7% to +2.4% for lipids in serum; and +0.4% to +6.2% for testosterone in serum and plasma from men and women. Spearman's rank correlation coefficients between measurements made in the baseline samples and those made after storage for 24 h were greater than 0.9 for 11 analytes, between 0.8 and 0.9 for 7 analytes, and less than 0.8 for only 1 analyte (vitamin C in serum). The ratio of between-subject to within-subject coefficients of variation was greater than 3.0 for all analytes except lutein (ratio, 1.6), alpha-cryptoxanthin (ratio, 2.4) and vitamin C (ratio in serum, 3.0; ratio in plasma, 2.2). We conclude that storage of whole blood at 4 degrees C for 24 h before freezing does not cause important changes in the analytes studied and that this delay in processing may be incorporated in the design of large prospective studies.