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Global warming appears to favour smaller-bodied organisms, but whether larger species are also more vulnerable to thermal extremes, as suggested for past mass-extinction events, is still an open question. Here, we tested whether interspecific differences in thermal tolerance (heat and cold) of ectotherm organisms are linked to differences in their body mass and genome size (as a proxy for cell size). Since the vulnerability of larger, aquatic taxa to warming has been attributed to the oxygen limitation hypothesis, we also assessed how body mass and genome size modulate thermal tolerance in species with contrasting breathing modes, habitats and life stages. A database with the upper (CTmax) and lower (CTmin) critical thermal limits and their methodological aspects was assembled comprising more than 500 species of ectotherms. Our results demonstrate that thermal tolerance in ectotherms is dependent on body mass and genome size and these relationships became especially evident in prolonged experimental trials where energy efficiency gains importance. During long-term trials, CTmax was impaired in larger-bodied water-breathers, consistent with a role for oxygen limitation. Variation in CTmin was mostly explained by the combined effects of body mass and genome size and it was enhanced in larger-celled, air-breathing species during long-term trials, consistent with a role for depolarization of cell membranes. Our results also highlight the importance of accounting for phylogeny and exposure duration. Especially when considering long-term trials, the observed effects on thermal limits are more in line with the warming-induced reduction in body mass observed during long-term rearing experiments.