Urban and transport planners need to assess the stressful conditions experienced by cyclists, considering that highly stressful situations can discourage people from cycling as a transport mode. Therefore, this study has two objectives: (1) to present a method for monitoring stress and other environmental factors along cycling routes using smart sensors; and (2) to analyze the influence of noise, vibration, presence of cycle paths, and the period of the day on stress experienced by cyclists. Data were collected in the city of São Carlos, Brazil, using stress and noise sensors, accelerometers, and Global Positioning System (GPS). Primarily, heat maps generated from the data made it possible to identify critical points of stress along the routes. In addition, the results of a logistic regression model were analyzed to identify the influence of the studied variables on stress. Although high levels of noise increased the odds of experiencing stress by 4%, very uncomfortable vibrations increased the odds by 14%, and the presence of cycle paths reduced the odds by 8%, an analysis of p-values and odds ratio confidence intervals shows, with a 95% confidence level, that only the period of the day influenced stress, as confirmed by the data. In this case, the odds of having stress increased by 24% in the afternoon rush hour compared to the morning rush hour.