Tuesday's protest follows a call by Catalan trade unions for a general strike in Spain's northeastern region to condemn police violence, which left nearly 900 injured, during Sunday's unauthorised vote. Barcelona's port was at a standstill.
But Catalan leaders claimed the results showed the region had the right to secede and said they may unilaterally declare independence.
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Giving a rare address to parliament, the royal called for calm but accused Barcelona of behaving "irresponsibly" by holding a referendum on independence which had been declared illegal by Madrid.
FC Barcelona said it would take part in the strike, adding that it would close its headquarters and that none of its professional or youth teams would train. However, despite crackdowns that ranged from the physical to the virtual, the referendum managed a turnout of 42.3% - and of those 2.2 million people who voted, just over 90% opted for Catalonian independence.
However, many services under the control of the Catalan government did see some stoppages, with public transport running at around 40 percent, according to reports, while port workers and civil servants also walked out. But given the chaotic nature of the vote, all figures should be taken with great caution.
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"Spain will maintain that this vote is not legitimate but the strength of feeling demonstrated can not be ignored by Spain, nor by the wider European and global community", said Fiona Hyslop, Scotland's minister for external affairs.
While the Socialist leader urged Mr Rajoy to hold talks with the Catalan president immediately, Mr Rivera said Spain should invoke article 155 of the constitution, in effect suspending Catalonia's autonomous powers.
Rajoy Monday also held meetings with Pedro Sanchez, the leader of the Spanish Socialist Party and Albert Rivera, the leader of center-right party Ciudadanos to discuss possible future action and government response to the crisis.
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But many teams chose not to protest at all including the Indianapolis Colts who had every member of the team standing. As the controversy entered its second week, echoes of support came from the president's fellow party leaders.
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