The seven-time Ballon d'Or winner has scored 11 goals and registered five assists in 12 games since arriving at Inter Miami this summer.
But the 36-year-old played just 37 minutes in the 4-0 win over Toronto FC on Wednesday before he was substituted due to injury.
Messi's minutes were already being managed as he enters the twilight of a spectacular career.
He was rested for Argentina's 3-0 win over Bolivia during the international break after scoring the winner against Ecuador a few days before.
His exploits for his country had already seen him miss two Miami fixtures - a 3-2 triumph over Sporting KC and a 5-2 hammering to Atlanta United.
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And now Messi's latest issue has sparked fears over his fitness ahead of next week's US Open Cup Final.
Is Messi fit to face Orlando City this weekend?
Messi will miss out on Inter Miami's next match against Orlando City, who are second in the MLS Eastern Conference.
"There is no chance they will be there on Sunday," boss Gerardo Martino told reporters on the availability of Messi and Jordi Alba.
"But I do not think there is anything serious or muscle injuries. I think it is just fatigue.
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"I know we have a final to play [on Wednesday] but they won't go near the pitch if they can't play.
"It wasn't part of the plan. I wouldn't say it was anything new or more serious than what was there before.
"I don't think there are any muscular injuries and we will take it day by day."
Martino added: "The scans [Messi] had with the national team, they didn't show any injury. We still saved him to be safe.
"We don't think he has a muscular injury. That's also from a conversation I just had with him.
"But we have to continue being careful. We'll look at it in the next few days. I'm cautious about what can happen and after talking to them, I'm less pessimistic than I was when I had to bring them off."
Inter Miami's caution surrounding star man Messi is not a surprise, with the David Beckham-led franchise set for a hectic upcoming schedule.
Following their derby with Florida rivals Orlando, Miami are involved in a US Open Cup Final clash with Houston Dynamo next week.
And then five league matches in 20 days follow for Messi and co against New York City, Chicago Fire, and Cincinnati FC as well as a double header with Charlotte FC.