This paper focuses on the effect of a sudden increase of plasma glucose concentration in the cardiac autonomic modulation using time-domain and frequency-domain heart rate variability (HRV) measures. Plasma glucose and insulin levels, measured each 30 min during an oral glucose tolerance test, and [Formula: see text] (mean of the RR interval), SDNN (standard deviation of normal-to-normal heartbeats), rMSSD (root-mean-square of successive differences between normal heartbeats), TP (total spectral power), LF and HF (power of the low- and high-frequency bands), LF norm and HF norm (LF and HF in normalized units), and LF/HF ratio of the HRV signal, obtained from 5-min-long ECG recordings during each phase of the test, were analyzed for subjects with the metabolic syndrome, marathon runners, and a control group. Results show that, after the glucose load, subjects with the metabolic syndrome experienced an increased sympathetic and decreased parasympathetic tone, which suggests an imbalance in cardiac autonomic modulation as a consequence of hyperglycemia and hyperinsulinemia. The significance of this study lies in the use of the ECG to assess the effects of a sudden increase in plasma glucose concentration on the cardiac autonomic modulation in subjects with different cardiovascular and metabolic conditions. Graphical Abstract Time-domain and frequency-domain heart rate variability measures are altered in subjects with different cardiovascular and metabolic conditions during an oral glucose tolerance test.