Cyclists are one of the main categories of road users particularly exposed to accident risk. The increasing use of this ecological means of transport requires a specific assessment of cyclist safety in terms of traffic flow and human factors. In this study, a particular visual tracking tool has been used to highlight not only the main critical points of the infrastructure, where a high level of distraction is recorded, but also the various interactions with different road users (pedestrians, vehicles, buses, wheelchairs, cyclists). To confirm the critical aspects of the infrastructure and the trend of workload, a similar circuit was reproduced in a bicycle simulator, which also allowed a meaningful comparison of cycling behaviour. The innovative component of this paper is a comparison between a real test, held in Stockholm, and a simulator where the same scenario has been represented, in order to highlight the objective differences in behaviour. The cycling performance was also evaluated both from an objective point of view, with the count of frames related to each category of visualization, and from a subjective one, through the questionnaires. The results show the crossing as a critical aspect because only 4/3% fixation is required for both simulated and real tests to confirm the significance of the comparison between the two experiments. The high attention rate, resulting from frame-by-frame analysis, also points to a clear difference in the perception of users, who feel with a low workload.