Differences in driving behavior due to the presence of users familiar/unfamiliar with the road are commonly considered in both road design (guidelines for road design) and traffic engineering (calculation of the equivalent traffic flow). However, although considered, the matter is largely unexplored: there is a lack of theoretical foundations and data on determining the impact of route familiarity on accident rates, speed choice and risk perception. On the other hand, some literature studies confirm that route familiarity is influential on driver behavior, mainly increasing inattention while driving, encouraging research in this sense. This paper reports the results of an on-road test carried out on a two lane rural road in the District of Bari in Puglia Region (Italy) over six days of testing, in order to find relationships between route familiarity, speed and trajectories selection, and risk perception. The particular schedule used for the tests allows to consider the influence of familiarity on the behavior of the test drivers. In particular, data were analyzed by considering the influence of road geometry and human factors.