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Introduction One in 4,000 infants is born with oesophageal atresia. Approximately 15% of these have a long gap oesophageal atresia, where primary anastomosis is difficult or impossible. Previous studies have shown an effect of intramural botulinum toxin type A (BTX-A) injections on the elongation and max load of the esophagus 1 hour after injection. We hypothesized that a longer waiting period of 2 hours could increase this effect. Methods Forty-five piglets were randomized into three groups. Two treatment groups received 2 units/kg of BTX-A and one group received saline. After 1 or 2 hours, a segment of the esophagus was harvested and put in a stretch-tension device to assess elongation and max load. Results Elongation from preload to max load and percentage elongation in the BTX-A 2h group (17.09 mm, 46.46%) was significantly higher compared with the BTX-A 1h group (13.59 mm, 40.16%) and the placebo group (13.77 mm, 39.92%). Conclusion Elongation of the piglet esophagus was significantly improved with a 2-hour waiting period after BTX-A injection. Injections with BTX-A could be useful in oesophageal atresia, where primary anastomosis is not possible. [ABSTRACT FROM AUTHOR]