Background: It is unknown how different types of carbohydrates alter the cardio-autonomic system in healthy individuals. Therefore, the aim of this study was to investigate how heart-rate variability changes to single dose ingestion of glucose, fructose, glucose and fructose, and an artificial sweetener (sucralose). Methods: In a double-blind randomized crossover placebo-controlled setting, 15 participants received all study-specific substances in liquid form. During each 2-h visit, venous blood glucose was measured in a 5-min interval while heart-rate variability was measured continuously via Holter-electrocardiograph. Results: Ingestion of different types of carbohydrates and sucralose showed significant differences for heart rate (p < 0.001), SDNN (p < 0.008), RMSSD (p < 0.001), pNN50 (p < 0.001) and blood pressure (p < 0.001). Different glucose levels significantly altered parameters of heart-rate variability and blood pressure (all p < 0.001), while the rate of change in blood glucose led to changes in heart rate variability, but not in heart rate (p = 0.25) or blood pressure (p = 0.99). Conclusions: Ingestion of different types of carbohydrates lead to reductions in heart-rate variability compared to a placebo. Blood glucose values above or below 70–90 mg/dL decreased heart rate variability while this was also seen for rapid glucose changes, yet not as pronounced. Healthy individuals should be conscious about carbohydrate intake while maintaining blood glucose levels between 70–90 mg/dL.