Messi was a late withdrawal for the Albiceleste, who will not risk their captain.
"I came here already feeling discomfort... I was training normally but on the last day I felt the discomfort and decided to 'clean up' everything for what is coming," Messi said prior to the game.
"I wanted to play, but as I said recently, there is still a long way to go. We have important things in the club, I hope it can be good to play with Spain to continue my recovery."
The captain is replaced by Giovani Lo Celso, who will occupy the playmaking role in Jorge Sampaoli's 4-2-3-1 formation.
Aguero too is a predictable absentee, with Argentina confirming his withdrawal from the squad minutes before kick-off.
The Man City star had trained with his international team-mates but was never expected to take the field in either of the two friendlies as he recovers from his own injury problem.
For Italy, meanwhile, veteran goalkeeper Gianluigi Buffon returns to the fray, while there are starts for PSG star Marco Verratti against club team-mates Angel Di Maria and Lo Celso as well as Lazio's Ciro Immobile.
Argentina: Caballero, Bustos, Otamendi, Fazio, Tagliafico, Paredes, Biglia, Lanzini, Lo Celso, Di Maria, Higuain.
Italy: Buffon; Florenzi, Bonucci, Rugani, De Sciglio; Parolo, Verratti, Jorginho; Chiesa, Immobile, Insigne.