BACKGROUND:This study assessed the acute effect of 21 days of challenging exercise on heart structure and function in recreationally active people across a range of age categories. METHODS:15 recreationally active people completed a 21-day fundraising cycling ride (MADRIDE) over a distance of 3515 km. Twenty-four hour Holter electrocardiography and blood biochemistry analyses were performed before and after the MADRIDE. RESULTS:Incidence of cardiac arrhythmia was higher after MADRIDE (OR: 5.93; 95% CI: 5.68-6.19), with increases in both ventricular arrhythmias (OR: 9.90; 95% CI: 9.27-10.57) and supraventricular arrhythmias (OR: 3.09; 95% CI: 2.91-3.29). Adults (OR: 11.45; 95% CI: 7.41-17.69) and older adults (OR: 10.42 95% CI 9.83-11.05) were approximately 10 times more likely to experience arrhythmias after the MADRIDE. Whereas, young participants experienced 18% less cardiac arrhythmias after MADRIDE (OR: 0.82; 95% CI: 0.75-0.90). Aortic valve max velocity was reduced (MD: -0.12 m/s; 95% CI: -0.19-0.05 m/s) and mitral valve deceleration time was slower (MD: -28.91 m/s; 95% CI: -50.97-6.84 m/s) after MADRIDE. Other structural and functional characteristics along with heart rate variability were not different after MADRIDE. CONCLUSIONS:Multi-day challenging exercise increased the incidence of both supraventricular and ventricular arrhythmias in active adults and older adults. Increases in arrhythmia rates after MADRIDE occurred without changes in cardiac structure and autonomic control. Further exploration is necessary to identify the causes of exercise-induced cardiac arrhythmia in adult and older adults.