The 36-year-old lasted just 37 minutes in Inter Miami's 4-0 MLS victory over Toronto, with the forward heading down the tunnel after being substituted.
He was later seen on the bench with his eyes closed as he laid back into the chair.
Inter are looking to win their second piece of silverware this season following their Leagues Cup success when they face Houston Dynamo in the US Open Cup on September 28.
But it remains to be seen whether Messi will be available, with Inter boss Gerardo Martino confirming he will miss the next match against Orlando.
As well as Messi, his ex-Barcelona teammate Jordi Alba also went off with an issue, as he stressed the pair were suffering from fatigue.
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Both players had already missed Inter's previous match - a 5-2 defeat away at Atlanta - due to fitness issues.
Martino said in his post-match press conference: "They trained normally and we felt like they were ready to play.
"I don't believe it's anything new or anything worse than what they had. It's fatigue. I don't think it's a muscular injury.
"There is no chance for them to play the Orlando match, but I do not think it's anything serious or muscle injuries. I think it is just fatigue."
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Messi's early exit means it is the first time since October 2018 that he has been withdrawn from play before half-time.
Incredibly, it is just the eighth time it has happened from his 937 career starts.
The 2022 World Cup winner has been nothing short of sensational since linking up with Inter on a free transfer.
In 11 games in all competitions, he has chipped in with 11 goals and five assists.
But with the win over Toronto being the first of six games in 18 days for Miami, Messi will have to be managed carefully.