Our aim was to identify proteins that reflect an acute systemic response to prolonged hyperbaric stress and discover potential biomarker pathways for pulmonary O₂ toxicity. The study was a double-blind, randomized, crossover design in trained male Navy diver subjects. Each subject completed two dry resting hyperbaric chamber dives separated by a minimum of one week. One dive exposed the subject to 6.5 h of 100% oxygen (O₂) at 2ATA. The alternate dive exposed the subjects to an enhanced air nitrox mixture (EAN) containing 30.6% O₂ at the same depth for the same duration. Venous blood samples collected before (PRE) and after (POST) each dive were prepared and submitted to LC-MS/MS analysis (2 h runs). A total of 346 total proteins were detected and analyzed. A total of 12 proteins were significantly increased at EANPOST (vs. EANPRE), including proteins in hemostasis and immune signaling and activation. Significantly increased proteins at O₂PRE (vs. O₂POST) included neural cell adhesion molecule 1, glycoprotein Ib, catalase, hemoglobin subunit beta, fibulin-like proteins, and complement proteins. EANPOST and O₂POST differed in biomarkers related to coagulation, immune signaling and activation, and metabolism. Of particular interest is (EANPOST vs. O₂POST), which is protective against oxidative stress.