Kyle Walker and Harry Maguire have withdrawn from the England camp with injuries, the English Football Association (FA) announced on Sunday.
The duo, along with goalkeeper Sam Johnstone, will return to their respective clubs for further assessment on injuries they picked up in recent days as per the FA.
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Walker, who was named England captain for the clash against Brazil on Saturday, was substituted in the 20th minute after appearing to injure his hamstring when tracking back to save a goal-bound shot off the line from Vinícius Júnior early in the game.
"We don't know [how serious it is]," England manager Gareth Southgate told reporters after the game, which Brazil won 1-0. "He's not had many injuries so he's not sure if it's just tightness or something more serious. We'll know more over the next few days."
Maguire lasted till 67 minutes at Wembley on Saturday, before being replaced by Lewis Dunk.
The latest spate of withdrawals serve a fresh blow to Southgate, who has already seen Harry Kane and Bukayo Saka pull out of the England squad in recent days.
Kane, who was in the stands for England's loss to Brazil on Saturday, had linked up with the squad ahead of the game despite picking up an ankle injury in Bayern Munich's win over Darmstardt before the international break.
Speaking after the game on Saturday, Southgate confirmed that the forward would be returning to his club to recover from his injury and will not be involved in England's friendly against Belgium on Tuesday.
Saka, who like Kane had joined the England setup ahead of this batch of friendlies, left the camp at St.George's Park last week after the FA announced he had arrived with an injury and had been unable to participate in training sessions.
Manchester City defender Rico Lewis, who made his debut against North Macedonia in November, and uncapped Burnley goalkeeper James Trafford have been moved up from the under-21 team to the senior squad.