Oxygen therapy has been widely used in lung injury (Li), adult respiraotory syndrome (ARDS) and inflammatory lung diseases as well as in mechanical ventilation in intensive care units. Exposure to hyperoxia is known to induct the production of reactive oxygen species (ROS) by mitochondria. Despite decades of research, the role of hyperoxia training in oxidative stress and ROS formation in the lungs is not known. The purpose of this study was to examine the effects of periodic-hyperoxia training on biological antioxidants (BAP) and lactate dehydrogenase (LDH) activities and free radicals (FR) production. Thirty adult male rats, matched with age and body weigh, were randomly assigned to three groups. The first group served as control (C) and the second (HP) was exposed to hyperoxia for 48. Animals in the third group (HP-T) were trained on hyperoxia for 1.5 h daily for three weeks. Following the exposure period for each group animals were sacrificed and lungs tissues were homogenized for BAP, LDH and FR determinations. LDH activity was determined by Randox protocol (Randox - UK). BAP and FR were determined using dROM method (H&D - Italy). Results showed that mean (±SD) BAP activity increased significantly (p < 0.05) from the baseline control of 7105.88 ± 2021.49 to 8611.20 ± 1245.26 (U/L) after hyperoxia training; then dropped to 6784.00 ± 1879.50 during hyperoxia exposure for 48 h. Whereas mean (±SD) FR production increased significantly (p < 0.05) from the baseline control of 262.50 ± 67.52 to 339.90 ± 64.84 during HP exposure for 48 h, then dropped to 211.13 ± 52.05 (Carr), during HP training. Similarly, LDH activity increased significantly (p < 0.05) from the baseline control of 210.31 ± 70.93 to 339.90 ± 64.84 during HP exposure for 48 h, then dropped to 159.30 ± 20.61(U/L), following HP-periodic training. Furthermore, the correlation (r = 0.67×) of LDH on FR was significant (p < 0.05), implying that reduction in ROS generation induced by HP-periodic training is related to reduced rate of cell apoptosis caused oxidative stress. Based on the results of the present study HP-periodic training is recommended in order to resist oxidative damage in the lungs.