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Background: Several studies have shown that adult patients with Hypoplastic Left Heart Syndrome (HLHS) and Fontan circulation have a reduced exercise tolerance that affects daily life. Recent studies have investigated the effects of aerobic exercise training in patients with univentricular heart; however, this research topic is still poorly studied. The aim of this study was to evaluate the effects of an aerobic exercise training program on cardiopulmonary exercise testing parameters and cardiac biomarkers in patients with HLHS. Methods: We enrolled 12 patients with a mean age of 24 ± 2.5 years (range 22–27 years), 50% male, with HLHS at Bambino Gesù Children’s Hospital IRCCS. All patients underwent a cardiopulmonary test and blood sampling before (T0) and after (T1) a 4-week aerobic exercise program. Cardiac biomarkers hs-cTnT, NT-proBNP, ST2, GDF-15 were studied. Results: Data analysis demonstrated an increase in cardiorespiratory performance after 4 weeks of aerobic exercise training activity. In particular, the data showed a significant improvement in test duration (p < 0.05), heart rate at rest (p < 0.05), heart rate recovery 1 min (p < 0.05), VO2 max (p < 0.01) and oxygen uptake efficiency slope (p < 0.05). At the same time, the data showed a significant reduction in NT-proBNP and ST2 values (p < 0.01 and p < 0.05, respectively) and a significant increase in GDF-15 (p < 0.01). No significant changes were found between the hs-cTnT values. Conclusions: Our study demonstrated the 4-week efficacy of an aerobic training program in improving cardiorespiratory performance and cardiac biomarker values in adult patients with HLHS and Fontan circulation. More studies with larger numbers of patients will be needed to confirm these data.