Argentina superstar Messi was substituted in the 73rd minute at a rain-soaked Nu Stadium in Miami after clutching the back of his left leg and requesting to come off.
The 38-year-old icon appeared to be walking normally as he headed straight down the tunnel after exiting the game, which Miami went on to win 6-4.
Inter Miami coach Guillermo Hoyos said after the game it was too early to determine if Messi was injured, and suggested the Argentine's substitution was precautionary due to the pitch conditions.
"As far as I know, we don't have a (medical) report on that yet, but we will have one shortly," Hoyos said after the game when asked for details of Messi's condition.
"He was genuinely suffering from fatigue in that regard - it is indeed fatigue. He was tired; the pitch was heavy, and when in doubt, the standard approach is always to ensure you don't take any risks."
Messi's Inter Miami team-mate Mateo Silvetti was unable to shed any light on his compatriot's condition.
"I'm just as in the dark as everyone else," Silvetti said. "At that moment, we were all focused on the match. I didn't know if it was an injury or if he was simply tired. We're hoping it turns out to be nothing serious. Given his mindset and everything else, we're going to wish him the very best."
Messi has carefully managed his workload since joining Inter Miami in 2023, with team staff regularly excusing him from games during particularly congested fixture periods.
He has spent multiple stints on the sidelines in recent years due to hamstring problems, which caused him to miss parts of Miami's pre-season campaign earlier this year.
Any significant injury to the eight-time Ballon D'Or winner, though, would deal a huge blow to Argentina's hopes of defending their World Cup crown in the United States, Canada, and Mexico.
The tournament kicks off in Mexico City on June 11th, with Argentina due to play their opening game against Algeria in Kansas City five days later.
Inter Miami's game on Sunday was the last before MLS takes a break ahead of the World Cup.
Messi has not formally confirmed he plans to play in the World Cup, but is widely expected to return for what will be a record-equalling sixth appearance at the finals.
Argentina's squad is due to be named next week, and the South American giants will head to the United States shortly afterwards for pre-World Cup friendlies against Honduras in Texas on June 6th, followed by a game against Iceland on June 9th.