ABSTRACT We investigated the effect of multiple freeze-thaw cycles on mumps, measles, and rubella virus serum antibody levels with whole-virus immunoglobulin G enzyme-linked immunoassays. Fresh serum samples from nine healthy adult volunteers were divided into six sets of five aliquots each. Samples were taken through a total of 10 freeze-thaw cycles and stored at 4°C until assayed. Each assay measurement was done in replicates of five, and the mean value was reported. After completing 10 freeze-thaw cycles, we found no clinically or statistically significant effect on measured antibody levels and found no discernible detrimental effect on the ability to measure these antibodies by enzyme-linked immunoassays.