Luke Shaw is losing his battle to be fit to return for England against Denmark on Thursday.
Gareth Southgate had earmarked the second group fixture as a game in which the Manchester United could feature.
But Shaw missed England training on Wednesday and instead continued his own fitness programme away from the rest of the team.
Shaw has not played since February due to a hamstring injury but Southgate hopes the only recognised left-back in the England squad will be ready to make a comeback soon.
With Shaw still battling back to fitness, Kieran Tripper is expected to continue at left-back against Denmark.
The other 25 players in the England squad trained on Wednesday.
Southgate will be tempted to continue his midfield experiment with Trent Alexander-Arnold against Denmark, as England seek a win that would secure qualification to the knockout stage.
Victory could even see them top the group depending on the result in the other game in the group between Serbia and Slovenia.
England finish their group campaign against Slovenia next week. Shaw will continue to be monitored by FA medical staff.