The impact of glucose and fructose supplementation on acute cardiac effects during cardiopulmonary exercise testing (CPET) is a topic that is rarely investigated. The aim of the presented secondary outcome analysis of a double-blind, randomized crossover-controlled trial was to investigate the impact of glucose (Glu), fructose (Fru), glucose and fructose (GluFru), and sucralose on electrocardiogram (ECG), heart rate variability (HRV), premature ventricular complexes (PVCs), and heart rate turn points (HRTP) during CPET. Fourteen healthy individuals (age 25.4 ± 2.5 years, body mass index (BMI) 23.7 ± 1.7 kg/m2, body mass (BM) of 76.3 ± 12.3 kg) participated in this study, of which 12 were included for analysis. Participants received 1 g/kg BM of Glu, 1 g/kg BM of Fru, 0.5 g/kg BM of GluFru (each), and 0.2 g sucralose dissolved in 300 mL 30 min prior to each exercise session. No relevant clinical pathology or significant inter-individual differences between our participants could be revealed for baseline ECG parameters, such as heart rate (HR) (mean HR 70 ± 16 bpm), PQ interval (146 ± 20 ms), QRS interval (87 ± 16 ms) and the QT (405 ± 39 ms), and QTc interval (431 ± 15 ms). We found preserved cardiac autonomic function by analyzing the acute effects of different Glu, Fru, GluFru, or sucralose supplementation on cardiac autonomic function by Schellong-1 testing. SDNN and RMSSD revealed normal sympathetic and parasympathetic activities displaying a balanced system of cardiac autonomic regulation across our participating subjects with no impact on the metabolism. During CPET performance analyses, HRV values did not indicate significant changes between the ingested drinks within the different time points. Comparing the HRTP of the CPET with endurance testing by variable metabolic conditions, no significant differences were found between the HRTP of the CPET data (170 ± 12 bpm), Glu (171 ± 10 bpm), Fru (171 ± 9 bpm), GluFru (172 ± 9 bpm), and sucralose (170 ± 8 bpm) (p = 0.83). Additionally, the obtained time to reach HRTP did not significantly differ between Glu (202 ± 75 s), Fru (190 ± 88 s), GluFru (210 ± 89 s), and sucralose (190 ± 34 s) (p = 0.59). The significance of this study lies in evaluating the varying metabolic conditions on cardiac autonomic modulation in young healthy individuals. In contrast, our participants showed comparable cardiac autonomic responses determined by ECG and CPET.