The 25-year-old strained his right calf during a workout session and the Blaugrana have decided not to risk the Argentine in the friendly at the Imtech Arena.
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Messi will now undergo physiotherapy to treat the injury and therefore the squad travelling to Hamburg for Tito Vilanova's first match in charge of Barcelona this season has been reduced to 21 players.
It had been reported that Hamburg will not pay Barcelona €1.2 million in appearance fees for the friendly as stipulated in the match agreement if Messi does not feature.
The game against Thorsten Fink's side is due to kick off at 18:00CET.