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Background Hemoglobin A1c (HbA1c) is an accurate index of fluctuation in glycemia over the 2-3 months prior to quantitative assessment. During this time, hemoglobin (Hb) slowly glycates until it shows the properties of advanced glycation end-products. Glycation kinetics is intensified by prolonged glucose exposure. In subjects undergoing oral glucose tolerance testing (OGTT), immediately after ingestion, glucose is ostensibly transported by the glucose transporter 1 (GLUT1) to erythrocyte corpuscular hemoglobin. The earliest significant measurable level of hemoglobin glycation associated with this transportation is still not clear. Subjects and methods We attempted to explore the early impact of short-term glucose load on HbA1c levels, because it is now known that transmembrane GLUT1-mediated glucose transport occurs immediately. A total of 88 participants (46 patients and 42 clinically healthy controls) underwent fasting plasma glucose quantitation during an OGTT. HbA1c, revealed by a monoclonal anti-glycation epitope antibody and adiponectin, was quantitated before (T0) and 2 hours (T120) after 80 g glucose ingestion. Results Wilcoxon test revealed that the HbA1c values did not significantly vary (P = 0.15) during the OGTT, whereas glucose concentration varied strongly between T0 and T120. Discussion It is well known that quantitative estimation of HbA1c is informative for clinical care, independently of glucose level. The molecular mechanisms and dynamics by which glucose enters/exits red blood cells are incompletely known and may differ between individuals. We here show, for the first time, that HbA1c levels do not significantly increase during OGTT, supporting the view that non-enzymatic glycation of hemoglobin occurs slowly and that glycation during the 2 hours of an OGTT is insignificant.