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Although HbA1c measurement by enzymatic assay (EA-HbA1c) is widely used in health-screening settings in Japan, recent studies have suggested lower EA-HbA1c levels as compared with HbA1c levels measured by high-performance liquid chromatography (HPLC-HbA1c). Hypothesizing that falsely low levels of EA-HbA1c are attributable to hemolysis caused by sample transport and/or storage, we measured EA-HbA1c in blood cells and whole blood after sample transport and compared them with HPLC-HbA1c levels. Blood samples were collected from ten non-diabetic individuals into sodium fluoride-containing blood collection tubes and immediately measured for EA-HbA1c in blood cells. After transport, the blood samples were again subjected to measurement of EA-HbA1c levels in blood cells and whole blood the following day. These EA-HbA1c levels were compared with HPLC-HbA1c levels. EA-HbA1c levels in blood cells measured immediately after sample collection did not significantly differ from HPLC-HbA1c levels. Transported blood samples showed hemolysis and significantly lower EA-HbA1c levels in blood cells, as compared with HPLC-HbA1c levels, whereas no significant difference was observed between EA-HbA1c levels in whole blood and HPLC-HbA1c levels. Transported blood samples showed hemolysis and falsely low EA-HbA1c levels in blood cells. Hemolysis caused by sample transport and/or storage might be responsible for the falsely low EA-HbA1c levels. This should be kept in mind, because falsely low HbA1c levels may lead to a false-negative diagnosis of diabetes.