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Neymar walked out of Brazil training, on Tuesday, in serious discomfort due to pain in his right ankle.
The Brazil striker was seen wincing and covered his face with a hand in distress, sparking fresh fears about his fitness.
He will spend the rest of the day undergoing physiotherapy, the Brazilian Football Confederation said.
Neymar recovered sufficiently from a broken foot to play in his side’s World Cup opener against Switzerland but is continuing rehabilitation on the injury.
He was accompanied off the pitch at Yug Sport Stadium in Sochi where he had been working with his Selecao team-mates.
But two members of the Brazil staff trudged off the pitch with the 26-year-old when he was unable to continue.
On Monday, Neymar had posted a picture of his foot receiving treatment alongside the message: ‘Working hard.’
Neymar broke his foot back in February while playing for the Parisians, but showed on social media that he is still receiving treatment to get it back up to 100 per cent.
PSG’s £198m man struggled to make his mark in the 1-1 draw against Switzerland and was dealt with physically by the opposition.
Neymar will expect similar treatment in Brazil’s next clash with Costa Rica on Friday if he plays but there now seems to be a question mark over his participation.