Brexit Party MEPs turned their backs during a rendition of Ode To Joy in the European Parliament as chaotic scenes marked the opening day of the new legislature in Strasbourg.
Leader Nigel Farage and newly elected candidates including Ann Widdecombe and Annunziata Rees-Mogg performed the protest as representatives gathered for the first session since May’s European elections.
Ukip’s MEPs – then led by Mr Farage – performed the same political stand at the start of the session in 2014.
Paul Nuttall, the party’s deputy leader at the time, said it was intended to send a message that they did not “recognise or respect the EU flag or anthem”.
Others in the parliament refused to stand at all as the EU’s anthem – composed by Ludwig van Beethoven in 1824 – was played by a jazz ensemble.
Reflecting the political divisions being felt over Brexit in the UK, Liberal Democrat MEPs took their seats wearing yellow “bollocks to Brexit” T-shirts.
Mr Farage told BBC Radio 4’s PM that the European Parliament president Antonio Tajani had told Brexit Party MEPs to stand up during the EU anthem.
“He said ‘look, you should stand up for an anthem even if it is of a foreign country’.
“So he was admitting that the European Union, as far as he’s concerned, is a country.
“So we obeyed his order and stood, but we decided to turn our backs.”
He went on to say: “What I object to is the way illegitimately these institutions have taken power and now have the nerve to call themselves a country.”
The Brexit Party and Lib Dems – each with opposing views on leaving the EU – came first and second respectively in the EU polls.
Meanwhile, newly-elected Green MEP Magid Magid, claimed he had been asked to leave the parliament building on his first day.
He tweeted: “I know I’m visibly different. I don’t have the privilege to hide my identity. I’m BLACK & my name is Magid. I don’t intend to try fit in. Get used to it!”
Mr Magid, who was wearing a baseball cap and t-shirt reading “f*** fascism”, told PA he was unsure who the official was, but added: “I make people feel uncomfortable, people don’t know how to react.”
The former Lord Mayor of Sheffield continued: “MEPs don’t reflect the people that they represent, Europe-wide and I know i’m gonna stick out like a sore thumb.”
A European Parliament spokeswoman said it had investigated the incident and confirmed that no Parliament staff were responsible, but was not able to say who was.
Protests were also held against the decision to deny a seat to Catalan separatist Carles Puigdemont and in defence of a German ship captain being held in Italy in a row over migrant rights.
Tuesday marks the opening of the new five-year session of the parliament, though the length of the UK’s involvement remains in doubt.
UK MEPs may sit in the parliament until the country formally leaves the EU.
A deadline of October 31 has currently been set for the UK to leave, though this could be extended if a deal is not found by then.