Background: Consistently successful patient outcomes following catheter ablation (CA) for treatment of persistent atrial fibrillation (pers-AF) remain elusive. We propose an electrocardiogram (ECG) analysis designed to (1) refine selection of patients most likely to benefit from ablation, and (2) examine the temporal evolution of AF organization indices that could act as clinical indicators of ongoing ablation effectiveness and completeness. Method: Twelve-lead ECG was continuously recorded in 40 patients (61 +/- 8 years) during stepwise CA (step-CA) procedures for treatment of pers-AF (sustained duration 19 +/- 11 months). Following standard pre-processing, ECG signals were divided into 10-s epochs and labeled according to their temporal placement: pre-PVI (baseline), dur-PVI (during pulmonary vein isolation), and post-PVI (during complex-fractionated atrial electrograms and linear ablation). Instantaneous frequency (IF), adaptive organization index (AOI), sample entropy (SampEn) and f-wave amplitude (FWA) measures were calculated and analyzed during each of the three temporal steps. Temporal evolution of these measures was assessed using a statistical test for mean value transitions, as an indicator of changes in AF organization. Results were then compared between: (i) patients grouped according to step-CA outcome; (ii) patients grouped according to type of arrhythmia recurrence following the procedure, if applicable; (iii) within the same patient group during the three different temporal steps. Results: Stepwise CA patient outcomes were as follows: (1) left-atrium (LA) terminated, not recurring (LTN, n = 8), (2) LA terminated, recurring (LTR, n = 20), and (3) not LA terminated, all recurring at follow-up (NLT, n = 12). Among the LTR and NLT patients, recurrence occurred as AF in seven patients and atrial tachycardia or atrial flutter (AT/AFL) in the remaining 25 patients. The ECG measures indicated the lowest level of organization in the NLT group for all ablation steps. The highest organization was observed in the LTN group, while the LTR group displayed an intermediate level of organization. Regarding time evolution of ECG measures in dur-PVI and post-PVI recordings, stepwise ablation led to increases in AF organization in most patients, with no significant differences between the LTN, LTR, and NLT groups. The median decrease in IF and increase in AOI were significantly greater in AT/AFL recurring patients than in AF recurring patients; however, changes in the SampEn and FWA parameters were not significantly different between types of recurrence. Conclusion: Noninvasive ECG measures, though unable to predict arrhythmia recurrence following ablation, show the lowest levels of AF organization in patients that do not respond well to step-CA. Increasing AF organization in post-PVI may be associated with organized arrhythmia recurrence after a single ablation procedure.