--- - |2+ Key Points Accumulating evidence indicates that passive heat therapy (chronic use of hot tubs or saunas) has widespread physiological benefits, including enhanced resistance against novel stressors (‘stress resistance’). Using a cell culture model to isolate the key stimuli that are likely to underlie physiological adaptation with heat therapy, we showed that both mild elevations in temperature (to 39°C) and exposure to serum from human subjects who have undergone 8 weeks of heat therapy (i.e. altered circulating factors) independently prevented oxidative and inflammatory stress associated with hypoxia‐reoxygenation in cultured endothelial cells. Our results elucidate some of the mechanisms (i.e. direct effects of temperature vs. circulating factors) by which heat therapy seems to improve resistance against oxidative and inflammatory stress. Heat therapy may be a promising intervention for reducing cellular damage following ischaemic events, which has broad implications for patients with cardiovascular diseases and conditions characterized by ‘chronic’ ischaemia (e.g. peripheral artery disease, metabolic diseases, obesity). Abstract Repeated exposure to passive heat stress (‘heat therapy’) has widespread physiological benefits, including cellular protection against novel stressors. Increased heat shock protein (HSP) expression and upregulation of circulating factors may impart this protection. We tested the isolated abilities of mild heat pretreatment and serum from human subjects (n = 10) who had undergone 8 weeks of heat therapy to protect against cellular stress following hypoxia‐reoxygenation (H/R), a model of ischaemic cardiovascular events. Cultured human umbilical vein endothelial cells were incubated for 24 h at 37°C (control), 39°C (heat pretreatment) or 37°C with 10% serum collected before and after 8 weeks of passive heat therapy (four to five times per week to increase rectal temperature to ≥ 38.5°C for 60 min). Cells were then collected before and after incubation at 1% O2 for 16 h (hypoxia; 37°C), followed by 20% O2 for 4 h (reoxygenation; 37°C) and assessed for markers of cell stress. In control cells, H/R increased nuclear NF‐κB p65 protein (i.e. activation) by 106 ± 38%, increased IL‐6 release by 37 ± 8% and increased superoxide production by 272 ± 45%. Both heat pretreatment and exposure to heat therapy serum prevented H/R‐induced NF‐κB activation and attenuated superoxide production; by contrast, only exposure to serum attenuated IL‐6 release. H/R also decreased cytoplasmic haemeoxygenase‐1 (HO‐1) protein (known to suppress NF‐κB), in control cells (−25 ± 8%), whereas HO‐1 protein increased following H/R in cells pretreated with heat or serum‐exposed, providing a possible mechanism of protection against H/R. These data indicate heat therapy is capable of imparting resistance against inflammatory and oxidative stress via direct heat and humoral factors. - The Journal of Physiology, Volume 596, Issue 20, Page 4831-4845, 15 October 2018.