Transportation infrastructure is one of the mainstays of human modification of terrestrial landscapes. Turtle populations are highly affected by roads through direct mortality, contributing to population declines. However, sub-lethal effects, such as increased physiological stress, may indirectly affect turtle demographic rates, particularly in populations recently exposed to roads. We took advantage of a unique study system in southeast Ohio, where an intact forest was bisected by a four-lane highway in 2013, exposing eastern box turtles (Terrapene carolina carolina) to a new threat. The goal of this study was to evaluate ecological, physiological, and behavioral effects of exposure to a new road by comparing a roadside turtle population to a control population in a nearby roadless area, and guide mitigation on new and existing roadways. We used a unique combination of radio telemetry to assess space use, behavior, and habitat selection of turtle, and bioassay techniques to analyze chronic stress using corticosterone stored in nail keratin. We found no differences in home range sizes and habitat selection between the two sites, but roadside turtles showed strong highway avoidance, despite spending a significant amount of time in its immediate vicinity. All turtles selected for higher woody debris and understory vegetation cover, and males at both sites selected for higher canopy cover. Corticosterone concentrations from nails collected upon initial capture (2017) did not differ between the two sites, but males showed a wider range of variation. Corticosterone concentrations were significantly higher in 2018, with roadside animals showing the highest levels, but they were not correlated with home range size or proximity to highway. As such, further work is needed to evaluate indirect effects of multiple stressors on turtle endocrinology and their demographic implications, as well as the level of demographic compensation resulting from road avoidance.