Scottish Deputy First Minister Nicola Sturgeon has said the political momentum is with the Yes campaign as the Glasgow Commonwealth Games come to a close, in an interview with a London-based Sunday newspaper.
But Mr Clegg said the Games should not be "distorted or sullied by the politics of the SNP" during a visit to Edinburgh yesterday.
He said sport can have a "galvanising effect on big political issues of the day" - citing the sporting boycott of Apartheid South Africa, black athlete Jesse Owen's Olympic triumph in Nazi Germany, and his own call for Russia to be stripped of the World Cup in 2018.
But he said on the whole "it is much better if politics and sport stay separate".
In her interview with The Observer, Ms Sturgeon said the Commonwealth Games would "inevitably leave a feelgood factor". She added: "I think confidence not only in Glasgow but across the country is high."
"I think there is a very significant momentum behind the Yes campaign and I feel it everywhere I go in the country. The momentum is with us and as we come out of the Commonwealth Games at the weekend that is us into the final strait of the campaign and that momentum will be visible."
Speaking at a training session of Murrayfield Wanderers in Edinburgh, Mr Clegg said: "I never for one minute thought that sport, and the fact that everyone can enjoy watching Commonwealth sport, was suddenly going to influence how people vote on the future of Scotland - whether it remains in the UK or not.
"I think it was very misplaced and gauche of Nicola Sturgeon this morning to try and extract political advantage from the Commonwealth Games.
"The Commonwealth Games are exactly what everybody wants them to be, which is a wonderful festival of sport bringing athletes together from around the Commonwealth, from around the world, and it really shouldn't be distorted or sullied by the politics of the SNP."
A spokesman for Nicola Sturgeon said: "Nick Clegg wouldn't get any medals for self-awareness, but maybe a gold for hypocrisy.
"While the No campaign shamelessly tried to politicise the Olympics, nothing that Nicola said about the Commonwealth Games reflects Nick Clegg's silly accusation, as any fair-minded reading of the interview will confirm.
"As she made clear in her Observer interview, Team Scotland has done incredibly well at the Commonwealth Games, winning a record number of medals, but sport is sport and politics is politics. For the Deputy Prime Minister to try and make political capital by twisting her comments shows just how worried the No campaign is by the opinion polls, which show that only a three per cent swing is needed for Scotland to achieve independence and all the opportunities that come with that."