Video report by ITV News Europe Editor James Mates
MEPs from the Brexit Party turned their backs while the European anthem played during the European Parliament’s opening session.
Nigel Farage and Ann Widdecombe were among MEPs, some of whom were in the European Parliament for the first time, to turn their backs while Ode To Joy played.
Brexit Party leader Nigel Farage took to Twitter to post that his new grouping had "already made its presence felt".
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”.
Reflecting the political divisions being felt over Brexit in the UK, Liberal Democrat MEPs took their seats wearing yellow “b******s 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.
Legislators from right-wing parties, as well as Catalan independence backers, also disrupted the formal opening session in Brussels, with some refusing to stand as a jazz ensemble played the anthem.
Prior to a musical quartet performing the anthem, the European Parliament's outgoing president Antonio Tajani had urged MEPs to show "respect" by standing for the piece.
He said: "Rising to your feet is a question of respect - it doesn't mean that you necessarily share the views of the EU.
"If you listen to the anthem of another country you rise to your feet."
As well as the members of the Brexit Party, Conservative MEP Geoffrey Van Orden also remained seated for the anthem.
There was also a protest against the decision to stop former Catalan president Carles Puigdemont taking his seat and a defence of the German ship captain who is held in Italy in a standoff over migrant rights.
After the formal opening, normal proceedings soon resumed.
Tuesday marks the first day in the new five-year session of the legislature following the May elections which set the scene for a more fractured parliament.
Away from the European Parliament, EU leaders appear to have finally made progress on identifying candidates for the bloc’s key posts.
Belgian Prime Minister Charles Michel has been nominated new European Council president - and would take over from Donald Tusk - while German Defence Minister Ursula von der Leyen is set to become new European Commission president, succeeding Jean-Claude Juncker.
And International Monetary Fund chief Christine Lagarde says she is giving up her IMF duties temporarily now that European Union leaders nominated her for the presidency of the European Central Bank.