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Introduction Hypobaric exposure reduces nitrogen solubility in body tissues, leading to the formation of microbubbles. Previous research has shown that a high-altitude environment significantly increases oxidative stress. This study aims to investigate the oxidative stress responses and associated factors in aircrew personnel undergoing hypobaric chamber training.Methods A longitudinal study design was employed to recruit 47 participants (13 females), comprising aircrew from the tri-service branches and inside observers from the Aviation Physiology Research Laboratory in Taiwan. Participants underwent the hypobaric chamber training profiles with maximum simulated altitudes of 25 000 or 35 000 feet (ft). Urine samples were collected at baseline, 30 min and 120 min post-training. Oxidative stress biomarkers assessed included 8-hydroxy-2′-deoxyguanosine (8-OHdG), malondialdehyde and Trolox equivalent antioxidant capacity (TEAC). Data analyses were performed using SPSS V.21.0.Results All biomarkers exhibited a similar temporal pattern, with an increase at 30 min followed by a decline at 120 min post-training. However, only the TEAC showed a statistically significant change, decreasing from 6.22±2.75 to 5.12±2.29 mmol/g creatinine (p=0.012). Females exhibited higher baseline 8-OHdG levels than males (2.40±1.29 vs 1.63±1.19 µg/mg creatinine; p=0.039), while male levels remained relatively stable. Participants exposed to a maximum simulated altitude of 35 000 ft demonstrated significantly lower post-training TEAC than those trained at 25 000 ft (5.70±2.51 vs 7.21±3.01 mmol/g creatinine; p=0.001). People who smoke exhibited higher 8-OHdG levels than people who do not smoke at 120 min post-training (p=0.010).Conclusions This non-invasive, longitudinal study demonstrated that hypobaric chamber training transiently alters oxidative stress biomarkers, particularly TEAC. Oxidative stress responses varied according to sex, training altitude, chamber position and smoking, highlighting the individual differences in the physiological adaptation to hypobaric exposure.