Marcelo is a doubt for Real Madrid's trip to Villarreal on Saturday after suffering a thigh injury during Brazil's 6-0 rout of Australia, while midfielder Isco is also struggling to recover from an injury problem.
Marcelo, 25, was withdrawn at half-time after complaining of soreness in his thigh, and the Brazilian football association confirmed he would not be involved in Tuesday's clash with Portugal.
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He has returned to Madrid, where club doctors are expected to assess the extent of his injury.
Isco has also created a problem for Real coach Carlo Ancelotti, with the 21-year-old continuing to struggle with the ankle injury he picked up last week.
The Spaniard was left out of his country's win over Finland on Friday and has also travelled back to Madrid for treatment after being ruled out of Spain's friendly with Chile on Tuesday.