The level of inspired oxygen during surgery may modify free radical release, and reperfusion injury. This controlled trial examined the effect of inspired oxygen on F2-isoprostanes (F2-IsoPs), isofurans (IsoFs) and specialised mediators of inflammation resolution (SPM) during knee replacement surgery. Patients received either 30% O2 (Control n = 21), 50% O2 (n = 20) or 80% O2 (n = 19) O2, in a parallel design. Haemoglobin (Hb) was measured throughout the surgery and F2-IsoPs, IsoFs and SPM were analysed by mass spectrometry. The effect of O2 on F2-IsoPs and IsoFs was examined during tourniquet inflation and after tourniquet release. SPM were measured at baseline and the end of surgery. There was a significant interaction between O2 and Hb concentrations with plasma IsoFs during tourniquet inflation. An increase in plasma IsoFs over time was attenuated in the 80% O2 group (P = 0.012) compared with the 30% O2 group after adjusting for Hb concentration. After tourniquet release, plasma F2-IsoPs were significantly lower in the 50% and 80% O2 groups (p = 0.009 and P = 0.001, respectively) compared with the 30% O2 group after adjustment for Hb concentration. The SPM RvD2 and RvE2 were increased with 50% and 80% O2 (RvD2, P = 0.014 and P = 0.002, respectively; RvE2, P = 0.032 with 50% O2) compared with the 30% O2 group, in analyses that corrected for Hb concentration.We have shown for the first time that higher O2 levels may be beneficial in reducing oxidative stress and increasing resolution of inflammation during surgery that involves reperfusion after application of a tourniquet.