Spain trio Carles Puyol, Santiago Cazorla and David Silva all failed to take part in training on Monday and are expected to miss the final Group D match against Greece.
Cazorla and Puyol have complained of sore feet while Silva twisted his ankle during training on Sunday.
Coach Luis Aragones has already hinted that he could make sweeping changes to his starting line-up against Greece, as victories over Sweden and Russia have guaranteed his side will finish in top spot in the group.
The veteran coach said: "I was going to say that after a couple more days with my head resting on my pillow I might change my idea about starting a completely fresh team against Greece.
"However, I'm going to have to stop blaming the pillow because I don't actually spend much time with my head on it.
"Not because of anything about the competition which is bothering me, just because I generally get very little sleep. That's just the way I am.
"There were 20 minutes against Sweden when the game was running away from us and we were stretched.
"But when we sorted it out in the second half I think that our stamina was better than theirs and all the running around they did in the first half caused them to tire.
"Seeing that, as I did against Russia too, was one of the most pleasing parts of the victory."