Making sense of heat tolerance estimates in ectotherms: lessons from<i>Drosophila</i>

<jats:title>Summary</jats:title><jats:p><jats:bold>1.</jats:bold> An increasing body of knowledge suggests that the estimation of critical upper thermal limits (CT<jats:sub>max</jats:sub>) is highly dependent on the experimental methodology employed. Here, we employ a theoretical approach to analyse how estimates of CT<jats:sub>max</jats:sub>(knock‐down temperatures and times) are affected by measurement protocol.</jats:p><jats:p><jats:bold>2.</jats:bold> Our model is able to reproduce the results of empirical studies on<jats:italic>Drosophila melanogaster</jats:italic>, suggesting that it adequately mimics organismal responses during assays. With simulated data sets, we also show that many experimental protocols result in unreliable and often highly biased estimates of CT<jats:sub>max</jats:sub>in<jats:italic>Drosophila</jats:italic>and possibly in other ectotherms.</jats:p><jats:p><jats:bold>3.</jats:bold> The confounding effects of stochasticity, resource depletion (or fatigue) and short‐term acclimatory responses are expected to be higher in longer assays, and therefore, short assays should be generally preferred. The experimental protocol of choice must also take into consideration the range in which measurement accuracy is not affected and the potential problems of thermal inertia in larger organisms.</jats:p><jats:p><jats:bold>4.</jats:bold> Our findings justify previous concerns that the methodology may have a greater impact on estimates of CT<jats:sub>max</jats:sub>than the biological process under study, and explain why many studies on the subject have often reported inconsistent and even contradictory results.</jats:p>