Kevin Sinfield will begin his attempt to run 101 miles in 24 hours to raise money for motor neurone disease research on Monday.
The route begins at Welford Road, where he is a coach for Leicester Tigers.
And it will end at the Headingley home of rugby league club Leeds Rhinos, where he played for 18 years.
His ex-Rhinos team-mate Rob Burrow was diagnosed with MND in 2019 and Sinfield raised £2.7m last year by running seven marathons in seven days.
The 41-year-old, who was made an OBE in the Queen’s Birthday Honours for fundraising and services to rugby league in June, set an initial target of £100,000 for the Extra Mile Challenge.
But he said last week: “We’re at about £70k with an extra £10k in gift aid and then there’s a significant amount in sponsorship, which takes us to about £150k.
“There’s one guy whose former business partner died of MND who has given almost £33,000 which is absolutely incredible.”
His route will be split into 7km sections and he must complete each one within 60 minutes.
Speaking to BBC Radio Leeds, Sinfield said he expected the experience would be “horrible and unpleasant”.
He continued: “A big part of the challenge is mental. There’s physical parts of it that are going to hurt and be horrible, but I think mentally every hour some of it is going to be torturous.”
Sinfield – who ran the London Marathon last month – will leave Welford Road at 08:40 GMT and aims to reach Nottingham by 14:40, where he will be joined on one leg by former Manchester City, Liverpool and England footballer Gemma Bonner.
“I’ve known Kev since we were seven or eight in school. I knew Rob too from that age so I have a long-standing connection with them. They were unbelievable role models for me,” she said.
“It’s devastating the situation that Rob is in, but it [the run] shows the people that are around him, what they are about and what they represent. I always aspire to be like them. I’m grateful to be a tiny help to raise awareness.”
Progressing north, Sinfield hopes to arrive in south Yorkshire around 21:00 on Monday and will continue throughout the night, with the final leg from Hyde Park in Leeds to Headingley beginning at 07:40 on Tuesday.
MND affects the brain and nerves and eventually stops muscles functioning.
The government has pledged to put at least £50m into MND research over the next five years.
Burrow hopes to be at the finishing line but Sinfield added: “Whether Rob is going to be good enough to be there on Tuesday, we’ll have to wait and see.
“If he is, I’ll be absolutely made up. If he’s not, I’ll understand. I’m sure there’ll be a couple of text messages en route where he’s laughing and taking the mickey out of us.
“But I absolutely know he will be with us every step of the way.”