European Commission chief Jean-Claude Juncker has said there would be no renegotiations with Britain following its membership referendum.
"The British policy makers and British voters have to know that there will not be any kind of renegotiation," Mr Juncker told reporters in Brussels.
"Out is out."
French president Francois Hollande said the future of the EU is at stake as voters in the UK decide tomorrow on whether to stay in or leave the union.
"It is the future of the European Union ... that is at stake," Mr Hollande said, warning that a Brexit would put Britain at "very serious risk" of losing access to the bloc's single market.
The two sides in the EU referendum campaign are making their final pitches ahead of polling day.
Tomorrow voters in the UK will be asked: "Should the United Kingdom remain a member of the European Union?"
With polls still claiming the race as neck-and-neck, campaigners are also trying to ensure that the 46 million people eligible to vote cast their ballot.
Much of the debate has boiled down to two issues: the economy and immigration.
The City of London, the International Monetary Fund and the majority of British business leaders back British Prime Minister David Cameron and his Remain camp's stance that to leave the EU would plunge Britain into recession, costing jobs and raising prices.
Those supporting a "Brexit" have struck a chord with many voters by saying Britain would regain control of immigration if it cut itself loose from a bloc they regard as domineering and out of touch.
Both camps have been accused of using unfounded assertions and scare tactics.
Highlights from last night’s #Brexit debatehttps://t.co/rcyEmFsMzR
— RTÉ News (@rtenews) June 22, 2016
In one of the last debates, London's newly-elected mayor Sadiq Khan accused Boris Johnson, the main leader of the Leave campaign, of exploiting fears of immigration to stoke anti-EU opinions.
"Your campaign hasn't been 'project fear'. It's been 'project hate' as far as immigration is concerned," Mr Khan said to huge applause from the 6,000 crowd at a live TV debate at London's Wembley Arena last night night.
Mr Johnson, favourite with bookmakers to replace Mr Cameron if Leave wins, told the cheering audience: "This Thursday can be our country's independence day."
Mr Johnson said the Remain camp spoke of nothing but fear and was "rubbishing" Britain.
He is travelling across Britain today in a helicopter to try to mobilise voters.
UK business chiefs warn over Brexit on referendum eve
Brexit in quotes
A guide to Brexit
Opinion polls have painted a contradictory picture of public opinion in a deeply divided nation.
But some published since last week's murder of Labour MP Jo Cox have suggested a slight lead for Remain, though often within the margin of error.
Today would have been Ms Cox's 42nd birthday. Events will take place to remember her across the UK as well as across the world.
Polling stations open at 7am tomorrow and close at 10pm.
The official result is due about 7am Friday but partial results and turnout figures from 382 counting centres will be announced through the night.
World leaders including US President Barack Obama, Chinese President Xi Jinping, German Chancellor Angela Merkel and the NATO and Commonwealth allies have urged Britain to remain in the EU.
