Cristiano Ronaldo, Pepe and Raul Meireles will all miss Portugal's 2014 World Cup warm-up match against Mexico in Boston on Friday, the Portuguese Football Federation (FPF) confirmed on Thursday.
"The three players are considered out of the match against Mexico," the FPF said in a statement.
Ronaldo has a special training regimen aimed at getting over leg injuries that threaten his participation in Portugal's World Cup opener.
Midfielder Raul Meireles is suffering from a right thigh injury, while defender Pepe did not take part in training at the grounds of the New York Jets on Thursday, as he undergoes physiotherapy on his left leg.
On Tuesday, the federation confirmed that Ronaldo, who was known to be nursing a left thigh injury, also has patellar tendinitis in his left knee.
The physical problems of their captain and talisman could deal an early blow to Portugal's World Cup bid as they face a tough first round in Group G, which also includes powerful Germany, Ghana and the United States.
Ronaldo, who has scored 49 goals in 110 matches for Portugal, didn't play in the country's first World Cup warm-up match, a goalless draw with Greece on Saturday in Lisbon.
Two more friendlies are on Portugal's slate before they depart the United States for Brazil, against Mexico on Friday and against Ireland on Tuesday.
They face a daunting World Cup opener on June 16 against Germany.