Harvey Elliott and Ryan Sessegnon have pulled out of the England Under-21 squad to face Kosovo.
The duo have returned to their clubs ahead of Tuesday's opening Euro 2023 qualifying match in Milton Keynes.
Aston Villa midfielder Aaron Ramsey has now been called up from the Under-19s.
A Football Association statement read: "Liverpool player Elliott reported for international duty with an injury sustained during his side's recent draw against Chelsea.
"Following further assessment, it has been decided that the 18-year-old is unlikely to play any part in the forthcoming fixture against Kosovo so has returned to his club.
"Tottenham Hotspur player Sessegnon has also returned to his club for further assessment after picking up a knock in training."
Tuesday's game is Lee Carsley's first match in charge after Friday's friendly in Romania was called off just hours before the Young Lions were due to travel to Bucharest.
Two Covid-19 tests from the squad and staff came back positive on Thursday and a decision was made to cancel the match.
