Hypoxic head and neck tumors respond poorly to radiotherapy and can be identified using gene expression profiles. However, it is unknown whether treatment outcome is driven by acute or chronic hypoxia. Gene expression data of 398 head and neck cancers was collected. Four clinical hypoxia profiles were compared to in vitro acute and chronic hypoxia profiles. Chronic and acute hypoxia profiles were tested for their association to outcome using Cox proportional hazard analyses. In an initial set of 224 patients, scores of the four clinical hypoxia profiles correlated with each other and with chronic hypoxia. However, the acute hypoxia profile showed a stronger association with local recurrence after chemoradiotherapy (p = 0.02; HR = 3.1) than the four clinical (chronic hypoxia) profiles (p = 0.2; HR = 0.9). An independent set of 174 patients confirmed that acute hypoxia is a stronger prognostic factor than chronic hypoxia for overall survival, progression-free survival, local and locoregional control. Multivariable analyses accounting for known prognostic factors substantiate this finding (p = 0.045; p = 0.042; p = 0.018 and p = 0.003, respectively). In conclusion, the four clinical hypoxia profiles are related to chronic hypoxia and not acute hypoxia. The acute hypoxia profile shows a stronger association with patient outcome and should be incorporated into existing prediction models.