The only thing that is physically challenging about the Acropolis Night Walk is the length (two to three miles), the climb (there is a fairly significant, if gradual, elevation gain), and the time of day (it’s dark!).
The entire route is paved. Navigating stairs is not required. Everything is wheel-chair accessible, so long as the grade is manageable. A couple of the “side trips” on the tour (e.g., the overlook of the Hephaestion and a quick visit to the Odeon of Herodes Atticus) require leaving the level path and may need to be skipped.