The Blaugrana talisman could miss his side's upcoming games after being replaced at halftime on Wednesday night.
A first Blaugrana goal for Neymar cancelled out David Villa's opener for Atleti in the first leg of the Spanish Super Cup on Wednesday, but Messi's withdrawal at the break marred the visitors' spirited fightback.
"Messi has been replaced as a precaution following some discomfort in the back of the left thigh," read a Tweet on the club's official Twitter stream during the game.
The Argentina international will now undergo further tests on Thursday which will reveal the full extent of his problem, with supporters no doubt hoping he hasn't suffered the same problem that kept him out of the end of last season.
Messi sustained a hamstring injury in the first leg of Barcelona's Champions League quarterfinal clash with Paris Saint-Germain.
He then appeared as a substitute in the second leg to inspire Barca to victory, but the 26-year-old only aggravated the injury further and sat out the final three games of the campaign with the Liga title already secured.