AimsTo establish a temporal safety window for cryoablation at minimal temperatures and to assess the electrophysiological and histological changes as a function of the application duration.Methods and resultsTwenty mini-pigs underwent AV nodal cryoablation at −80°C without prior cryomapping. The duration of the cryoapplication following atrioventricular block (AVB) was randomized to 0, 10, 20, 40, or 60 s. Atrioventricular block was obtained in all animals after a median of 3 (1–8 interquartile range) applications. One week later, AV nodal conduction fully recovered in animals with application duration <10 s, whereas persistent AVB incidence increased as a function of time in animals with longer applications duration. Cryoablation application duration following AVB was the only independent predictor of persistent AVB (OR, 1.116; 95% CI, 1.013–1.229; P = 0.026). There was no difference in lesion location or size between animals with vs. those without persistent AVB at 1 week. However, animals randomized to longer application duration demonstrated higher degree of cell destruction and fibrotic content.ConclusionIn this closed-chest pig model, there was a relation between cryoapplication duration following AVB at −80°C and recovery of conduction. A safety window of at least 10 s was observed in all cases.