The 48-year-old was running in the Mighty Deerstalker event in the Scottish Borders.
But just one mile into the race the woman fell ill at the Innerleithen Bridge and emergency crews were called to the scene around 6pm on Saturday.
She died en route to Borders General Hospital.
An eye-witness told Metro.co.uk: ‘When I arrived she was lying there with people around her.
‘She was by a river that people have to run through – it was at calf height. Someone told me that she fainted at the water.
‘They put up a screen as they were doing CPR and everyone had to go past while it was happening, so it was a bit alarming for them.’
The race took off on Saturday in the Scottish Borders (Picture: Helen Barrington)
The run, which is billed as the UK’s biggest night race, was being held at Innerleithen in Tweeddale.
Participants must run through rivers, forests and over mountains in the pitch dark, wearing head-torches.
The woman’s name and the cause of death has not yet been released to the public.
A spokesman for organisers Rat Race Adventure Sports, said: ‘We are extremely saddened to confirm that a participant in our Mighty Deerstalker event passed away yesterday.
‘The participant became unwell at 5.56 pm, approximately one mile into the event, close to our event control station and at the location of one of our safety marshals.
‘A member of our medical team was on the scene within three minutes as we implemented our response protocols. They were assisted by an off-duty paramedic until the arrival of the Scottish Ambulance Service.
‘Tragically, we have since learned that the individual passed away en route to hospital.
‘Our heartfelt thoughts and condolences are with the family and friends of the deceased.’
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