Most available exposure-response relationships for assessing crop loss due to elevated ozone (O(3)) have been established using data from chamber and open-top chamber experiments, using a simulated constant O(3) concentration exposure (square wave), which is not consistent with the diurnal variation of O(3) concentration that occurs in nature. We investigated the response of oilseed rape (Brassica napus L.) to O(3) as affected by two exposure regimes: one with a diurnal variation (CF100D) and another with a constant concentration (CF100). Although the two exposure regimes have the same mean O(3) concentration and accumulated O(3) concentration above 40 ppb (AOT40), our results show that O(3) at CF100D reduced biomass and number of pods/plant more than O(3) at CF100. Both O(3) exposures resulted in larger seed weights/100 pods compared to CF. Numbers of seeds/100 pods were reduced by CF100, while numbers of seeds/100 pods in the CF100D chambers were comparable to those in CF. Our results suggest that chamber experiments that use a constant O(3) exposure may underestimate O(3) effects on biomass and yields.