Oxygen supply to the heart has been hypothesized to limit cardiac performance and whole animal acute thermal tolerance (CT_max) in fish. We tested these hypotheses by continuously measuring venous oxygen tension (P_VO2) and cardiovascular variables in vivo during acute warming in European perch (Perca fluviatilis) from a reference area during summer (18°C) and a chronically heated area (Biotest enclosure) that receives warm effluent water from a nuclear power plant and is normally 5-10°C above ambient (24°C at the time of experiments). While CT_max was 2.2°C higher in Biotest compared to reference perch, the peaks in cardiac output and heart rate prior to CT_max occurred at statistically similar P_VO2 values (2.3 - 4.0 kPa), suggesting that cardiac failure occurred at a common critical P_VO2 threshold. Environmental hyperoxia (200% air saturation) increased P_VO2 across temperatures in reference fish, but heart rate still declined at a similar temperature. CT_max of reference fish increased slightly (by 0.9°C) in hyperoxia, but remained significantly lower than in Biotest fish despite an improved cardiac output due to an elevated stroke volume. Thus, while cardiac oxygen supply appears critical to elevate stroke volume at high temperatures, oxygen limitation may not explain the bradycardia and arrhythmia that occur prior to CT_max. Acute thermal tolerance and its thermal plasticity can therefore only be partially attributed to cardiac failure from myocardial oxygen limitations, and likely involves limiting factors on multiple organizational levels.