Aims To present a comparison of electrocardiogram-based non-invasive measures of atrial fibrillation (AF) substrate complexity computed on invasive animal recordings to discriminate between short-term and long-term AF. The final objective is the selection of an optimal sub-set of measures for AF complexity assessment. Methods and results High-density epicardial direct contact mapping recordings (234 leads) were acquired from the right and the left atria of 17 goats in which AF was induced for 3 weeks (short-term AF group, N = 10) and 6 months (long-term AF group, N = 7). Several non-invasive measures of AF organization proposed in the literature in the last decade were investigated to assess their power in discriminating between the short-term and long-term group. The best performing measures were identified, which when combined attained a correct classification rate of 100%. Their ability to predict standard invasive AF complexity measures was also tested, showing an average R-2 of 0.73 +/- 0.04. Conclusion An optimal set of measures of the AF substrate complexity was identified out of the set of non-invasive measures analysed in this study. These measures may contribute to improve patient-tailored diagnosis and therapy of sustained AF.