A high glycemic status increases the risk for autonomic dysfunction and cardiovascular failure. The aim of this study was to investigate time-dependent changes in the autonomic response and cardiovascular dynamics and the association between the level of hemoglobin A1c (HbA1c) and autonomic response during blood volume reduction. The study population consisted of 26 preoperative participants who were scheduled for autologous blood donation (200-400 mL of whole blood) for intraoperative or postoperative use. These participants without circulatory, respiratory, or brain disease and diabetes mellitus were grouped according to their HbA1c levels: < 6.5% (n = 18) and ≥ 6.5% (n = 8). We measured blood pressure (BP) and analyzed heart rate variability (HRV) to quantify cardiac autonomic regulation throughout blood donation. During blood volume reduction, which was about 10% of the circulating blood volume, the BP and heart rate varied within normal ranges in both groups. The high-frequency (HF) component, an index of parasympathetic nerve activity, and the ratio of low-frequency (LF) to HF components (LF/HF), an index of sympathetic nerve activity, significantly decreased and increased with the progression of blood volume reduction, respectively, in the HbA1c < 6.5% group. In contrast, in the HbA1c ≥ 6.5% group, the HF component did not significantly change, and the increase in the LF/HF ratio was delayed. Time-dependent changes in HRV were related to blood volume reduction only in the HbA1c < 6.5% group. Thus, elevated HbA1c levels are associated with the decrease in the autonomic response induced by blood volume reduction.