PURPOSE: Delivery of radiation at ultrahigh dose rates, known as FLASH, has recently been shown to preferentially spare normal tissues from radiation damage compared to tumor tissues. However, the underlying mechanism of this phenomenon remains unknown, with one of the most widely considered hypothesis being that the effect is related to substantial oxygen depletion upon FLASH, thereby altering the radiochemical damage during irradiation, leading to different radiation responses of normal and tumor cells. Testing of this hypothesis would be advanced by direct measurement of tissue oxygen in vivo during and after FLASH irradiation. METHODS AND MATERIALS: Oxygen measurements were performed in vitro and in vivo using the phosphorescence quenching method and a water-soluble molecular probe Oxyphor 2P. The changes in oxygen per unit dose (G-values) were quantified in response to irradiation by 10 MeV electron beam at either ultra-high dose rates (UHDR) reaching 300 Gy/s or conventional radiotherapy dose rates of 0.1 Gy/s. RESULTS: In vitro experiments with 5% BSA solutions at 23°C resulted in G-values for oxygen consumption of 0.19–0.21 mmHg/Gy (0.34–0.37 μM/Gy) for conventional irradiation and 0.16–0.17 mmHg/Gy (0.28–0.30 μM/Gy) for UHDR irradiation. In vivo, the total decrease in oxygen after a single fraction of 20 Gy FLASH irradiation was 2.3±0.3 mmHg in normal tissue and 1.0±0.2 mmHg in tumor tissue with p-value < 0.00001, while no decrease in oxygen was observed from a single fraction of 20 Gy applied in conventional mode. CONCLUSIONS: Our observations suggest that oxygen depletion to radiologically relevant levels of hypoxia is unlikely to occur in bulk tissue under FLASH irradiation. For the same dose FLASH irradiation induces less oxygen consumption than conventional irradiation in vitro, which may be related to the FLASH sparing effect. However, the difference in oxygen depletion between FLASH and conventional irradiation could not be quantified in vivo, because measurements of oxygen depletion under conventional irradiation are hampered by resupply of oxygen from the blood.