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Background: Peroral endoscopic pyloromyotomy is a novel technique that has recently been described in the literature. There is little data to guide the length of myotomy created. The aim of study was to evaluate the proper incision length of the muscular layer during peroral endoscopic pyloromyotomy using a submucosal tunnel technique. Methods: The study was designed as a prospective ex vivo study. Fresh ex vivo porcine stomachs from animals weighing 80-100 kg and porcine stomachs from animals weighing 15-25 kg were used for pyloromyotomy. Four different myotomy lengths (1, 2, 3, and 4) were compared in the large animal series and three different myotomy lengths (1, 2, and 3) were compared in the small series. A total of 23 cases of the submucosal tunnel technique were performed by two endoscopists using 12 large stomachs and 11 small stomachs. Results: The mean overall procedure time (±SD) of pyloromyotomy was 65.7 (±14.3) min. In the large stomach series, the mean pyloric diameter (±SD) and change from baseline (as percentage) following a 1, 2, 3, and 4 pyloromyotomy were 13.3 ± 9.5 mm (7.1 %), 20.7 ± 11.7 mm (10.6 %), 31.1 ± 15.0 mm (15.2 %), and 33.0 ± 15.0 mm (16.0 %), respectively. In the small stomach series, the changes of mean pyloric diameter following a 1, 2, and 3 cm pyloromyotomy were 12.2 ± 5.6 mm (7.5 %), 23.1 ± 7.6 mm (13.1 %), and 28.0 ± 10.4 mm (15.5 %), respectively. Conclusions: A 3 cm pyloromyotomy for a large animal series and 2 cm for the small animal series appeared to be most appropriate for enlargement of the pylorus. [ABSTRACT FROM AUTHOR]