Tayside Police yesterday launched an appeal for witnesses after the latest bid to halt the charity Etape Caledonia race in Highland Perthshire.
The tacks, along with drawing pins and staples, were scattered across a stretch of the route but were discovered by police before the race started.
Organisers also found about 30 drawing pins on the route on Saturday.
Yesterday extra road sweeping vehicles were brought in to clear the road after the second batch of sharp objects was found at about 3am. The event began at 6.45am as planned.
Amy Whitely, event director, said: "Following the incident on Saturday we were on high alert and were able to identify and deal with issues on the route quickly and without any disruption to the event.
"Participants' safety is our primary concern and we would urge any members of the local community with information in relation to this incident to contact Tayside Police."
A Tayside Police spokesman said: "A number of tacks, drawing pins, metal screws and staples were spread across the road at the event between Weem and Strathtay.
"The items were discovered prior to the start of the cycling event.
"One cyclist who was not connected to the event suffered a puncture. The event itself was not disrupted in any way.
"This attempt to disrupt the event was reckless and completely irresponsible and it is fortunate that no injuries were caused.
"Tayside Police will work closely with the organisers and the local community to find those responsible and to ensure the safety and security of all persons in the area."
"Anyone with any information who may have seen persons acting suspiciously along the Strathtay to Weem part of the Etape route or anyone who has any other information to assist should contact Tayside Police on 0300 111 2222, or pass information anonymously via the charity Crimestoppers on 0800 555 111."
Nearly 5,000 people took part, with riders including Graeme Obree, dubbed The Flying Scotsman. Before the race began, he dedicated the event to the memory of Wouter Weylandt, the professional Belgian cyclist who died following an accident during last week's Giro d'Italia.
A similar attempt to wreck the Etape – which has attracted criticism from a number of locals because it closes roads in the area – was made two years ago.
Several hundred cyclists had their tyres punctured during the event as they rode over the tacks which were spread over a stretch of road lasting about five miles.
Some cyclists at the front of the ride, hurtling at speeds of up to 40mph down the tack-strewn section of the road, fell badly, but there were no serious injuries.
Three cyclists in this year's event collided in an accident on the Tummel Bridge to Aberfeldy road, at 10:40am, with two taken to Dundee's Ninewell's Hospital with non-life threatening injuries. The road was closed while the accident was cleared.
The 81-mile Etape Caledonia race is the largest closed road cycling event in the UK.