The actors - better known as Carl King, Nikhil Sharma, John Barton and Andy Sugden on the ITV1 soap - are undertaking the 224-mile challenge in a bid to raise £100,000 for Macmillan Cancer Support.
Joined by the show's executive producer Steve November and professional cyclists, the cast members will set off from The Woolpack in Emmerdale Village tomorrow evening at 6pm, before cycling continuously throughout the night. They are aiming to arrive at EastEnders' iconic Queen Vic pub on Saturday evening.
Upon arrival, the participants will be greeted by members of the EastEnders cast who will congratulate them on their achievement.
The Emmerdale team are carrying out the feat in memory of the soap's late series producer Gavin Blyth, who passed away last year after losing a battle with cancer.
Sophie Gill, Macmillan's Community Corporate Manager, said: "The Macmillan Emmerdale to EastEnders cycle challenge is a fantastic tribute to Gavin, and another amazing example of how far the fab Emmerdale guys will go to help Macmillan Cancer Support and people affected by cancer. We hope Emmerdale fans and Macmillan supporters along the route will dig deep and help the team towards their £100,000 target."
Donations for the Emmerdale to EastEnders challenge can be made via a dedicated JustGiving page. Alternatively, a £5 donation can be made by texting 'RIDE66 £5' to 70070.
Updates on the project can be found on a special 'Team Emmerdale' website and video page.
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Watch a preview of tonight's Emmerdale double bill in the video below: