The Manchester United forward limped out of Tuesday's session and may miss England's game against Italy.
Danny Welbeck's fitness is a major concern for Roy Hodgson heading into England's first game of the World Cup against Italy on Saturday.
With Alex Oxlade-Chamberlain already sidelined for the opener with inflamed knee ligaments, England may be short of wide players after Welbeck pulled up in a team shape drill on Tuesday.
After missing training on Wednesday, Welbeck is in a race against time to be fit for the Italy game. Hodgson will be left with just Raheem Sterling, Adam Lallana and Wayne Rooney as options on the wing if the Manchester United star misses out.
However, Wayne Rooney has backed his club teammate to be fit for Saturday, saying "he will be fine."
There was some good injury news for England, as Steven Gerrard has recovered from a minor injury scare and will be fit to face Cesare Prandelli's side. The England captain played a full part in the training session on Tuesday.
Hodgson's side appeared to be in a relaxed mood on the eve of the tournament. The Wednesday training session ended with a game of heads, shoulders, knees and toes, a stark contrast from the strict, businesslike demeanor of the team under Fabio Capello at the 2010 World Cup in South Africa.
England will fly from Rio de Janeiro to Manaus on Thursday, ready for the Group D opener at the weekend.