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.
The Brexit Party and Lib Dems – each with opposing views on leaving the EU – came first and second respectively in the EU polls.
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.