Background: Airplane Headache (AH) occurs during flights and often appears as an intense, short lasting headache during take-off or landing. Reports are limited on pathological mechanisms underlying the occurrence of this headache. Proper diagnosis and treatments would benefit from identification of potential pathways involved in AH pathogenesis. This study aimed at providing a simulated airplane headache condition as a proxy towards identification of its underlying mechanisms. Methods: Fourteen participants including 7 volunteers suffering from AH and 7 healthy matched controls were recruited after meeting the diagnostic and safety criteria based on an approved study protocol. Simulation of AH was achieved by entering a pressure chamber with similar characteristics of an airplane flight. Selected potential biomarkers including salivary prostaglandin E-2 (PGE(2)), cortisol, facial thermo-images, blood pressure, pulse, and saturation pulse oxygen (SPO) were defined and values were collected before, during and after flight simulation in the pressure chamber. Salivary samples were analyzed with ELISA techniques, while data analysis and statistical tests were handled with SPSS version 22.0. Results: All participants in the AH-group experienced a headache attack similar to AH experience during flight. The non-AH-group did not experience any headaches. Our data showed that the values for PGE2, cortisol and SPO were significantly different in the AH-group in comparison with the non-AH-group during the flight simulation in the pressure chamber. Conclusion: The pressure chamber proved useful not only to provoke AH-like attack but also to study potential biomarkers for AH in this study. PGE(2), and cortisol levels together with SPO presented dysregulation during the simulated AH-attack in affected individuals compared with healthy controls. Based on these findings we propose to use pressure chamber as a model to induce AH, and thus assess new potential biomarkers for AH in future studies.