Ecological relevance and repeatability of results obtained in different laboratories are key issues when assessing thermal tolerance of ectotherms. Traditionally, assays have used acute exposures to extreme temperatures. The outcomes of ecologically more relevant ramping experiments, however, are dependent on the rate of temperature change leading to uncertainty of the causal factor for loss of function. Here, we test the physiological consequences of exposing female Drosophila melanogaster to gradually increasing temperatures in so-called ramping assays. We exposed flies to ramping at rates of 0.06 and 0.1°C min(-1), respectively. Flies were sampled from the two treatments at 28, 30, 32, 34, 36 and 38°C and tested for heat tolerance and expression levels of the heat shock genes hsp23 and hsp70, as well as Hsp70 protein. Heat shock genes were upregulated more with a slow compared with a faster ramping rate, and heat knock-down tolerance was higher in flies exposed to the faster rate. The fact that slow ramping induces a stronger stress response (Hsp expression) compared with faster ramping suggests that slow ramping induces more heat damage at the cellular level due to longer exposure time. This is supported by the observation that fast ramped flies have higher heat knock-down tolerance. Thus we observed both accumulation of thermal damage at the molecular level and heat hardening at the phenotypic level as a consequence of heat exposure. The balance between these processes is dependent on ramping rate leading to the observed variation in thermal tolerance when using different rates.