AbstractCells need to preserve genome integrity despite varying cellular and physical states. p53, the guardian of the genome, plays a crucial role in the cellular response to DNA damage by triggering cell cycle arrest, apoptosis or senescence. Mutations in p53 or alterations in its regulatory network are major driving forces in tumorigenesis. As multiple studies indicate beneficial effects for hyperthermic treatments during radiation- or chemotherapy of human cancers, we aimed to understand how p53 dynamics after genotoxic stress are modulated by changes in temperature across a physiological relevant range. To this end, we employed a combination of time-resolved live-cell microscopy and computational analysis techniques to characterise the p53 response in thousands of individual cells. Our results demonstrate that p53 dynamics upon ionizing radiation are temperature dependent. In the range of 33 °C to 39 °C, pulsatile p53 dynamics are modulated in their frequency. Above 40 °C, which corresponds to mild hyperthermia in a clinical setting, we observed a reversible phase transition towards sustained hyperaccumulation of p53 disrupting its canonical response to DNA double strand breaks. Moreover, we provide evidence that mild hyperthermia alone is sufficient to induce a p53 response in the absence of genotoxic stress. These insights highlight how the p53-mediated DNA damage response is affected by alterations in the physical state of a cell and how this can be exploited by appropriate timing of combination therapies to increase the efficiency of cancer treatments.