The Argentina international was brought off midway through the first half with a hamstring/thigh problem as his recent fitness issues persist.
The Argentine, 26, started in the central role for Gerardo Martino's men in the trip to the bottom-of-the-table Liga side but only lasted 21 minutes - the shortest he has ever featured for the club after being picked in the starting lineup.
Andres Iniesta was brought on in Messi's place, with the four-time Ballon d'Or hobbling off the pitch with no confirmation yet regarding how long the attacker will be out for. He is set to undergo additional tests Monday after inital scans indicated an injury to the femoral biceps of the player's left leg.
Messi broke Gerd Muller's record for goals scored in a calendar year in the fixture last season but has now gone five Liga games without a goal.
He is now a major doubt for Argentina's friendlies in the upcoming international window against Ecuador and Bosnia & Herzegovina.