PO₂ cycling, often referred to as intermittent hypoxia, involves exposing tissues to brief cycles of low oxygen environments immediately followed by hyperoxic conditions. After experiencing long-term hypoxia, muscle can be damaged during the subsequent reintroduction of oxygen, which leads to muscle dysfunction via reperfusion injury. The protective effect and mechanism behind PO₂ cycling in skeletal muscle during reoxygenation have yet to be fully elucidated. We hypothesize that PO₂ cycling effectively increases muscle fatigue resistance through reactive oxygen species (ROS), protein kinase B (Akt), extracellular signal-regulated kinase (ERK), and certain mitochondrial channels during reoxygenation. Using a dihydrofluorescein fluorescent probe, we detected the production of ROS in mouse diaphragmatic skeletal muscle in real time under confocal microscopy. Muscles treated with PO₂ cycling displayed significantly attenuated ROS levels (n = 5; p < 0.001) as well as enhanced force generation when compared to controls during reperfusion (n = 7; p < 0.05). We also used inhibitors for signaling molecules or membrane channels such as ROS, protein kinase B (Akt), extracellular signal-regulated kinase (ERK), as well as chemical stimulators to close mitochondrial ATP-sensitive potassium channel (KATP) or open mitochondrial permeability transition pore (mPTP). All these blockers or stimulators abolished improved muscle function with PO₂ cycling treatment. This current investigation has discovered a correlation between KATP and mPTP and the PO₂ cycling pathway in diaphragmatic skeletal muscle. Thus, we have identified a unique signaling pathway which may involve ROS, Akt, ERK and mitochondrial channels responsible for PO₂ cycling protection during reoxygenation conditions in the diaphragm.