The use of repellents is a unique measure of personal protection, which can avoid tick attachment and thus reduce the risk of tick-borne infections. In the European Union, the efficacy of the repellents coming onto the market has to be evaluated according to the guidelines published by the European Chemical Agency before registration. The United States Environmental Protection Agency has a similar role. Despite obvious differences in morphology and behavior, both these guidelines allow the use of nymph or adult female ticks for laboratory testing. Here, we provide evidence that sensitivity to diethyltoluamide (deet) ( < 0.0001) of nymphs within the in vitro trial was significantly higher than in adult females. In the experiment, we also observed that feral ticks were less sensitive to repellent than were laboratory-reared ticks ( < 0.01) and that mobility decreased when the trial was repeated ( < 0.05). This study showed that the results of efficacy tests could vary significantly even when the protocol was conducted in accordance with the recommended methods. To refine the results of efficacy tests, we recommend a reevaluation of the guidelines, with emphasis on the developmental stage and origin of ticks.