STUDY DESIGN: Cross-sectional study. OBJECTIVES: To compare cardiac autonomic modulation of individuals with spinal cord injury (SCI) that practice different amounts of moderate to vigorous physical activity (PA) and able-bodied controls at rest and during a non-immersive Virtual Reality task. SETTING: Athletes with SCI of wheelchair basketball, wheelchair tennis, wheelchair handball, WCMX (wheelchair motocross), and para-swimming were assessed at the Faca na Cadeira Institute, ICEL and Clube Esperia in Sao Paulo, Brazil; non-athletes with SCI and able-bodied controls were assessed at the Acreditando Centro de Recuperacao Neuromotora, Sao Paulo, Brazil. METHODS: One-hundred forty-five individuals were assessed: 36 athletes with traumatic SCI (41.1 +/- 16.8 years old), 52 non-athletes with traumatic SCI (40.2 +/- 14.1 years old), and 57 able-bodied individuals (39.4 +/- 12.5 years old). Cardiac autonomic modulation was assessed through heart rate variability (HRV) measured in the sitting position at rest and during a VR game activity. RESULTS: We found significantly more favourable HRV for athletes with SCI when compared to non-athletes with SCI, but no differences between athletes with SCI and able-bodied controls. In addition, athletes and able-bodied controls showed adequate autonomic nervous system (ANS) adaptation (rest versus physical activity in VR), i.e., they experienced parasympathetic withdrawal during VR physical activity, which was not found in non-athletes with SCI. CONCLUSION: The practice of moderate to vigorous physical activity is associated with healthier cardiac autonomic modulation in adults with SCI, which may lead to more favourable health outcomes. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04618003, retrospectively registered.