High altitude is the most intriguing natural laboratory to study human physiological response to hypoxic conditions. In this study, we investigated changes in reactive oxygen species (ROS) and oxidative stress biomarkers during exposure to hypobaric hypoxia in 16 lowlanders. Moreover, we looked at the potential relationship between ROS related cellular damage and optic nerve sheath diameter (ONSD) as an indirect measurement of intracranial pressure. Baseline measurement of clinical signs and symptoms, biological samples and ultrasonography were assessed at 262 m and after passive ascent to 3830 m (9, 24 and 72 h). After 24 h the imbalance between ROS production (+141%) and scavenging (−41%) reflected an increase in oxidative stress related damage of 50–85%. ONSD concurrently increased, but regression analysis did not infer a causal relationship between oxidative stress biomarkers and changes in ONSD. These results provide new insight regarding ROS homeostasis and potential pathophysiological mechanisms of acute exposure to hypobaric hypoxia, plus other disease states associated with oxidative-stress damage as a result of tissue hypoxia.