BACKGROUND: Previous work examining ethanol's autonomic effects has found contrasting patterns of age-related differences in ethanol-induced hypothermia between adolescent and adult rats. Most studies have found adolescents to be less sensitive than adults to this effect, although other work has indicated that adolescents may be more sensitive than adults under certain testing conditions. To test the hypothesis that adolescents show more ethanol hypothermia than adults when the amount of disruption induced by the test procedures is low, but less hypothermia when the experimental perturbation is greater, the present study examined the consequences of manipulating the amount of perturbation at the time of testing on ethanol-induced hypothermia in adolescent and adult rats. METHODS: The amount of test disruption was manipulated by administering ethanol through a chronically indwelling gastric cannula (low perturbation) versus via intragastric intubation (higher perturbation) in Experiment 1 or by either familiarizing animals to the handling and injection procedure for several days pretest or leaving them unmanipulated before testing in Experiment 2. RESULTS: The results showed that the handling manipulation, but not the use of gastric cannulae, altered the expression of ethanol-induced hypothermia differentially across age. When using a familiarization protocol sufficient to reduce the corticosterone response to the handling and injection procedure associated with testing, adolescents showed greater hypothermia than adults. In contrast, the opposite pattern of age differences in hypothermia was evident in animals that were not manipulated before the test day. Surprisingly, however, this difference across testing circumstances was driven by a marked reduction in hypothermia among adults who had been handled before testing, with handling having relatively little impact on ethanol hypothermia among adolescents. CONCLUSIONS: Observed differences between adolescents and adults in the autonomic consequences of ethanol were dramatically influenced by whether animals were familiarized with the handling/injection process before testing. Under these circumstances, adolescents were less susceptible than adults to the impact of experimental perturbation on ethanol-induced hypothermia. These findings suggest that seemingly innocuous aspects of experimental design can influence conclusions reached on ontogenetic differences in sensitivity to ethanol, at least when indexed by ethanol-induced hypothermia.