Portugal will take on Mexico on Saturday and Ronaldo (knee and thigh), Pepe (calf) and Meireles (thigh), who have been restricted to lighter training duties, will miss out again, having also failed to take part against Greece on May 31.
The trio will have just one more opportunity to gain match practice ahead of their World Cup opener against Germany on June 16 with Portugal set to play the Republic of Ireland on June 10.
"Cristiano Ronaldo, Pepe and Raul Meireles maintained a pre-established program by the medical department for their injuries," a statement from the Portuguese Football Federation (FPF) said on Thursday.
"The three players are considered unfit for the match with Mexico."
Portugal will clash with Mexico at Gillette Stadium in Foxborough, Massachusetts, while Paulo Bento's men will face the Republic of Ireland in New Jersey.
After the World Cup fixture against Germany in Salvador, Portugal will play the United States in Manaus on June 22 and Ghana in Brasilia four days later.
