Around 100 students blocked a motorway close to Turin, forcing police to re-route traffic. Another group surrounded the area where work on the rail link is expected to start in the Val di Susa region in the Alps.
Workers are preparing to dig an exploratory tunnel in a four-year project.
Authorities have taken over a total of seven hectares (17 acres) under a scheme in which the owners of the land are paid rent during the duration of the project and will then be returned their property at the end.
The total cost of the planned link is €20 billion and would shorten the journey from Paris to Milan to just four hours compared to seven hours now.
It has sparked opposition among some local residents who say it is too costly and could damage the environment, while supporters say it is a strategic project for Europe that will reduce road traffic by 700,000 trucks a year.
The rail link is currently expected to be completed in 2023.