They have also expressed their fears over the possible gridlock that may be caused on one of Scotland's most dangerous roads.
The event is due to take place on the A82 from Crianlarich to Fort Augustus in two stages on September 12 and 13. It is part of the Deloitte Ride Across Britain, billed as the UK's ultimate cycle challenge, over nine days.
Stewart MacLean of the A82 Partnership, which is campainging for the upgrading of the route from Glasgow to Inverness via Fort William said: "Having discussed the logistics of 800 cyclists setting off from Hamilton over a period of one to two hours with organisers I can only anticipate gridlock."
He said cars would be stuck behind the riders by the time they reach Onich, near Fort William, adding there were "no overtaking opportunities" between the village and the town.
The businessman added: "The original idea was to set them off in groups of 50 in three minute intervals. So you can imagine a block of 50 cyclists multiplied by about 15."
Lochaber councillor Brian Murphy was concerned about the cyclists' safety, adding: "The road struggles at any time of the year to cope with existing cycle traffic."
A Lochaber businessman, who did not wish to be named, said: "I'm sure I was caught up in this or something similar a couple of years ago between Onich and Fort William. It was very slow and hazardous drive trying to weave in and out between large, long groups of cyclists who often spread out quite far across and along the carriageway."
The organisers said the police, council and roads department had been advised of what was to come at least six weeks in advance and that they were proud of their safety record.
A Transport Scotland spokeswoman said: "We have been working alongside partners such as Police Scotland to provide advice on trunk road safety to the organisers of the proposed event."