Captain Makoto Hasebe (knee) and Gotoku Sakai (knee) are both expected to miss Japan's final warm-up match against Zambia at Raymond James Stadium in Florida on Friday.
Shinji Okazaki, Japan's leading scorer during World Cup qualifying with eight goals, is another doubt for the game after falling ill in the past few days.
Star duo Keisuke Honda and Shinji Kagawa have also been working their way back to full fitness but Zaccheroni says he is not losing sleep ahead of their World Cup opener against Group C opponents Ivory Coast in Brazil on June 14.
"We try to stick to our plan even though so far we had some hiccups relating to some players' physical condition," the Italian told reporters.
"That's because when you play in official games you are likely to get some knocks and concussions that affect your training.
"This is the last friendly before the World Cup. I will try to let some players play more minutes.
"There will be less planned substitutions."
Zaccheroni added: "The atmosphere at the camp is very positive and this is one of my objectives so that we face what's to come at our best."
One player seemingly free of injury and well placed to feature in the World Cup opener is Atsuto Uchida.
The fullback was struck down by a thigh injury during the 2013-14 Bundesliga campaign for Schalke but he has featured in Japan's past two games, scoring the unlikely winner against Cyprus on May 27.
"After playing 70 minutes, I don't have any setback," he said.
"When I get on a pitch, get hit or kicked by opponents, and start having wounds and feeling pain, I feel like 'I am now back playing'. It makes me feel like I am actually back in the game and I am happy.
"I can feel that it's getting better as I play. It's important to get used to winning before going into the tournament.
"Some of it will go away and we get back to a start line, but the mental preparation before matches, momentum and confidence, those things will be carried over and won't go away."
Japan will also face Greece (June 19) and Colombia (June 24) in Group C at the showpiece event.