The race, along Hadrian's Wall, starts at Carlisle Castle and ends at Newcastle's Quayside.
Its 69 mile (111km) route includes off-road and on-road terrain, and passes a number of tourist attractions such as Lanercost Priory and the market town of Hexham.
Organiser Jim Mee said it was "not for the faint-hearted".
Mr Mee, who is taking part in the race, said: "It's 69 miles across Hadrian's Wall country, which is pretty lumpy in the middle, so it's some undertaking.
"People see these crazy challenges out there in fantastic places and think 'yeah, I'd like a pop at that'."
Up to 1,000 people have set off on what organisers have described as the UK's largest non-stop ultra-marathon.