England Under 21 trio Michael Johnson, Jack Rodwell and Danny Welbeck have all been forced to withdraw from the squad through injury.
Stuart Pearce's men face Portugal in their European Championship qualifier at Wembley - all be it with a reduced capacity - on Saturday.
Johnson was due to win his first cap for 15 months after an abdominal injury restricted him to just eight appearances for Manchester City last season, but has picked up a knock.
Rodwell (groin) and Welbeck (knee) have also returned to their clubs, with Pearce calling up Everton's Dan Gosling and Arsenal's Henri Lansbury, who is currently on loan at Championship side Watford, in their place.
Despite the withdrawals, Pearce remains upbeat and highlights that playing at Wembley will be an invaluable experience for his young players.
"While most of them won't know yet what it's like to play a senior international match, at least they can get a feel of the stadium, the crowd, and the importance of needing to win the match," he said.
"It'll be a great atmosphere with over 30,000 people in the ground, our biggest crowd in two-and-half years since we played Italy at Wembley.
"It shows that people want to come and watch the U21s, but also come to this stadium and see a live match here at ticket prices that are fantastic for everyone to afford."