As a healthier and more sustainable way of mobility, cycling has been advocated by literature and policy. However, current trends in bicyclist crash fatalities suggest deficiencies in current roadway design in protecting these vulnerable road users. The lack of cycling data is a common challenge for studying bicyclists' safety, behavior, and comfort levels under different design contexts. To understand bicyclists' behavioral and physiological responses in an efficient and safe way, this study uses a bicycle simulator within an immersive virtual environment (IVE). Off-the-shelf sensors are utilized to evaluate bicyclists' cycling performance (speed and lane position) and physiological responses (eye tracking and heart rate (HR)). Participants bike in a simulated virtual environment modeled to scale from a real-world street with a shared bike lane (sharrow) to evaluate how introduction of a bike lane and a protected bike lane with pylons may impact perceptions of safety, as well as behavioral and psycho-physiological responses. Results from 50 participants show that the protected bike lane design received the highest perceived safety rating and exhibited the lowest average cycling speed. Furthermore, both the bike lane and the protected bike lane scenarios show a less dispersed gaze distribution than the as-built sharrow scenario, reflecting a higher gaze focus among bicyclists on the biking task in the bike lane and protected bike lane scenarios, compared to when bicyclists share right of way with vehicles. Additionally, heart rate change point results from the study suggest that creating dedicated zones for bicyclists (bike lanes or protected bike lanes) has the potential to reduce bicyclists' stress levels.