Sproles' latest setback comes after coach Doug Pederson said he expected the running back to play Sunday in his first game since Week 1.
Sources said Sproles was highly disappointed by the injury recurrence, as were the Eagles.
Sproles hurt the hamstring in practice leading into Week 2 and has missed the past seven games.
The Eagles had hoped Sproles could resume punt-return duties and rejoin a backfield currently being led by Wendell Smallwood and Corey Clement. Philadelphia ranks 18th in rushing yards per game this season with 106.6.
DeAndre Carter had been returning punts in Sproles' absence but was waived Tuesday and claimed by the Houston Texans on Wednesday.