Rhian Brewster, Eberechi Eze and Marc Guehi have withdrawn from England's U21 squad to face Albania as a precaution against injury.
Chelsea defender Guehi, who is currently on loan at Swansea, and Crystal Palace midfielder Eze returned to their respective clubs on Monday, while Sheffield United forward Brewster left the squad on Sunday.
It comes after England U21 manager Aidy Boothroyd said he would never risk players' fitness.
Boothroyd is already without Ryan Sessegnon, who withdrew from the squad prior to Friday's 3-1 win over Andorra.
Tottenham defender Sessegnon entered quarantine at the request of his loan club Hoffenheim, after a number of positive coronavirus cases at the German club.
England U21s face Albania at Molineux on Tuesday and Boothroyd has promised to not put players at unnecessary risk, with qualification for Euro 2021 already secured.
"They are injuries we have to monitor. If there was a cup final tomorrow all three would play but such is the nature of this season and the loading of the players it's the right thing to do for those guys not to take any risks," said Boothroyd.
"It's the right thing to do in order to get the players right for those clubs. The players are the most important.
"We have to work together to get the loading right. We want to be performing well and winning but it's important the players are fit and healthy."
Aaron Ramsdale will be rested against Albania with Stoke's Josef Bursik, on loan at Doncaster, starting instead for the Young Lions.
However, Boothroyd insists the Sheffield United goalkeeper remains his first-choice number one.
He said: "Joe is going to start the game but I spoke to Aaron at length and let me be clear he is on our number one, on and off the pitch. He is a brilliant guy to have around.
"He has had a bit of a tough time at the minute but I'm sure him and his club will turn the corner.
"He has got exactly the right character, he is going to be a top goalkeeper and we are really lucky to have him."