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 line-up.
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.
It is also uncertain as to what the injury is, with suggestions varying from a knock to a muscular problem, which follow on from his fitness issues in October.
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.