Led by coach Stefano Pioli, the Fiorentina squad gathered at the gates of the Artemio Franchi stadium where the team play their home games and placed a picture of Astori alongside the banners, scarves and messages previously left by fans.
They then stood in a semi-circle and applauded.
The club said in a statement that they would take part in a training session in the afternoon.
Astori, who made 289 Serie A appearances in 10 seasons and played 14 times for the national team, was found dead in his room at the team hotel in Udine where Fiorentina had been due to play Udinese on Sunday.
That match was postponed along with all other Serie A games.
"In honour of Davide Astori's memory and to make his memory indelible, Cagliari and Fiorentina have decided to retire the No. 13 shirt," Fiorentina said on Twitter. Cagliari posted a similar message.
The municipal government of Florence has declared a day of mourning for Thursday when Astori's funeral will take place. Schools and businesses have been asked to observe one minute's silence at 1300 local time (1200 GMT).
(Reporting by Shrivathsa Sridhar in Bengaluru and Brian Homewood in Zurich, editing by Ed Osmond and Christian Radnedge)
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