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Patients undergoing catheter ablation for atrial fibrillation (AF) frequently require redo procedures, but there are no data reporting interatrial septum thickness (IAS) and difficulty during repeat transseptal puncture (TSP).Patients undergoing two separate AF ablation procedures had preprocedural fossa ovalis (FO) thickness measured using transesophageal echocardiography (TEE). “Difficult” TSP was defined by two observers as requiring excessive force, or conversion to TEE guidance.The study comprised 42 patients (37 male) with mean ± SD age 55 ± 9 years. Mean FO thickness was significantly greater at the time of redo TSP (2.2 ± 1.6 mm vs 2.6 ± 1.5 mm at redo, P = 0.03); however, this finding was limited to those who underwent initial dual transseptal sheath procedures, FO thickness 2.0 ± 1.5 mm and 2.5 ± 1.4 mm for TEE 1 and 2, respectively (P = 0.048). There was a trend for more frequent difficult redo TSP procedures, 7/42 (17%; 95% confidence interval [CI] 8–31) redo, versus 4/42 (10%; 95% CI 3–23) first TSP. On univariate analysis, FO thickness was not predictive of TSP difficulty; the only predictor of difficult redo TSP was diabetes.IAS thickness at the FO increased following catheter ablation of AF, yet on subgroup analysis this was limited to initial procedures utilizing dual transseptal sheaths. There was a trend toward more frequent difficulty during redo TSP, yet this was not associated with FO thickening. Diabetes may predispose to difficulty during redo TSP; this finding requires confirmation in a larger study population.