NHS Dumfries and Galloway said it followed the endurance race at Drumlanrig Castle on 17 and 18 June.
The health board that events on farm land “inevitably” carried a small risk of gastrointestinal infection.
It advised anyone suffering from symptoms such as diarrhoea, vomiting or fever to seek medical advice.
The health board said people would usually notice any symptoms three to four days after they had been infected.
It added they could start up to a fortnight later.
A spokesman for Tough Mudder said safety for participants, volunteers and staff was a its “number one priority”.
“Tough Mudder is aware of the situation in Scotland and is working closely with local authorities,” he said.
“We are sorry to hear that Tough Mudder participants are unwell and wish them a full and speedy recovery.”