Barcelona forward Messi has not yet shaken off a thigh strain, while AC Milan ace Balotelli has a knee injury sustained in training.
Messi missed Barca’s final pre-season game against a Malaysia XI on Saturday and it has now been confirmed he will return to Catalunya to work on his recovery.
A statement on the club’s website read: “Leo Messi will not feature in the international friendly between Argentina and Italy in Rome this Wednesday.
“After undergoing tests conducted by the Argentina national team doctors, it was decided that Messi should return to Barcelona and focus on recovering from his muscle injury.
“The injury forced the forward miss out on Barca’s last match of the Asia Tour.”
Balotelli’s withdrawal was confirmed by the Italian FA on their website on Tuesday evening.
Meanwhile, Italy head coach Cesare Prandelli feels he might be distracted from the football on display during Wednesday’s clash in Rome – as he scans the Stadio Olimpico stands for a special guest.
The Azzurri meet the Argentinians – with whom they have shared many classic encounters in recent decades – for the first time in 12 years in a special fixture organised to welcome newly-elected Pope Francis, who is a Buenos Aires native.
Serie A favourite Hernan Crespo scored the winner as the Albiceleste won 2-1 in Rome in February 2001 and the visitors triumphed on penalties in a World Cup semi-final held in Naples 11 years previously.
Argentina and Italy were in the same group at the 1986 World Cup, too, but the South Americans went on to claim the trophy that Enzo Bearzot’s men had lifted in Spain four years before.
Prandelli believes both squads are united by their common respect for the evening’s special guest.
He told FIGC.com: “We play games every day, week in week out, but you only get to meet the Pope once in a lifetime. So I do not agree with Balotelli.
“The match against Argentina has special meaning because it is our welcome for Pope Francis. The two teams are staying in the same hotel and the atmosphere has to be one of a party spirit for everybody.
“I hope the Pope comes to watch the game. I don’t know if he’ll be able to come, but I hope he will. You don’t ask anything from the Holy Father, you just have to listen to him because he knows everything.
“We want to build some continuity from the hard work we did at the Confederations Cup and show that kind of balance through the entire game. That would represent a success.”
With Balotelli ruled out, Roma striker Pablo Osvaldo looks set to make his return to the starting line-up, alongside Alberto Gilardino, following an absence enforced by ill-discipline.
The Italo-Argentine forward fell foul of Prandelli’s code of conduct when he publicly insulted former Giallorossi boss Aurelio Andreazzoli in the wake of a Coppa Italia defeat to Lazio in May.
Question marks still hang over his future in the capital but Osvaldo is now back in the Italy fold and impressing his coach with his enthusiasm in training.
“I saw a determined Osvaldo and we even made a few jokes about what has happened,” Prandelli added.
“Does he have to stay in Rome? I don’t ever meddle in these matters, I can only say that the ideal thing would be for him to find some sort of balance in his life.
“I think he’s an important player for Roma but he needs to be in a calm environment if he’s to play at his best.”
Argentina may have lost the services of Messi, but they have selected star players including Gonzalo Higuain and Angel di Maria while coach Alejandro Sabella will hope to build some momentum before World Cup qualifying resumes against Paraguay next month.