Jules Kounde suffered a relapse while Eric Garcia was subbed off as Barcelona continued to face injury problems at their defensive line with just 20 days to the World Cup.
France centre-back Kounde suffered what appeared to be a hamstring injury in Barcelona's 1-0 win at Valencia on Saturday in LaLiga.
The defender, 23, was replaced by Gerard Pique and adds to defending champions France's list of mounting injury concerns ahead of the tournament in Qatar.
While Spanish defender Garcia was also forced off with a groin issue in the game, while Valencia's Uruguayan striker Edinson Cavani was taken off after appearing to aggravate an ankle problem.
Barcelona coach Xavi told DAZN TV that Kounde would undergo checks but at first sight "it seems like discomfort".
Chelsea's French midfielder N'Golo Kante has been ruled out of the tournament because of a hamstring injury, while Juventus' Paul Pogba is still a doubt after knee surgery.
Manchester United centre-back Raphael Varane suffered a hamstring injury last weekend, while goalkeepers Mike Maignan and Steve Mandanda have also suffered injuries, with the former unlikely to make the tournament.
Earlier, Spain striker Alvaro Morata was substituted by Atletico Madrid at Cadiz in the 10th minute after his foot was stepped on and he could not continue.
The World Cup starts on November 20, with France's first game against Australia on November 22.