The Liverpool captain and Manchester City forward will now return to their clubs for further assessment.
An official England statement read: “Jordan Henderson and Raheem Sterling will miss England’s forthcoming UEFA Nations League fixture against Iceland.
“The pair have returned to their respective clubs for further assessment. Gareth Southgate will continue his preparations for Wednesday with a 22-man squad.”
Sterling picked up a knock in training last week and missed the 2-0 defeat to Belgium, while Henderson started the game in Leuven before coming off at half-time for Harry Winks.
Gareth Southgate described Sterling’s issue as a “niggle” when discussing the player’s absence ahead of the Belgium match.
“Raheem felt something a couple of days ago, didn’t train on Friday as a precaution,” Southgate said.
“He started training yesterday (Saturday) and wasn’t really happy with it. So we’ve discussed it, it’s not one we should take a chance on. I think he’ll be fine for the end of the week, but the games for us will just come too quickly.
“It was a slightly heavy pitch when we trained on Friday and it is a small niggle really,” he continued.
“He had a good rest at the beginning of the week – it’s a bit of an unfortunate incident but not one that is going to keep him out for long.”
The news will be a concern for Pep Guardiola and Jurgen Klopp ahead of a pivotal weekend in the Premier League.
City face Tottenham on Saturday in a 5:30pm kick-off, while the champions take on Leicester City on Sunday in a 7:15pm kick-off.