On Thursday night against the Jets, starting center Khaled Holmes went down on the first series with an apparent ankle injury and rookie tackle Ulrick John left later with a lower right leg injury.
Both were scheduled for MRIs on Friday, but there was no immediate update from the team.
Defensive lineman Jeris Pendleton (knee) and linebacker Daniel Adongo (biceps) also left the game and were put on the waived-injured list Friday night. If they clear waivers, they will wind up on Indy's injured reserve list.
The biggest problem is along the offensive line, where the Colts already have lost left guard Donald Thomas for the season because of a torn right quad.
His injury put the Colts in the precarious position of going with what could be one of the youngest interior lines in the league - a rookie and two second-year players, including Holmes.
''Yeah we've got our work cut out for us,'' Pagano said after the 13-10 loss. ''We'll get home and we'll assess the damage, see the extent of those injuries and make the necessary moves that we have to make.''
The challenges are popping up everywhere.
In just two weeks, the Colts have lost Thomas and running back Vick Ballard (torn left Achilles tendon) with season-ending injuries.
Defensive end Fili Moala, who was expected to be a solid contributor this season, didn't even make it to training camp before he went on injured reserve with a season-ending knee injury. Indy's top fullback, Stanley Havili, is still on the physically unable to perform list following offseason shoulder surgery.
Indy held out Pro Bowl receiver Reggie Wayne and running back Ahmad Bradshaw as a precaution Thursday after they finished last season on injured reserve.
The Colts also played Thursday without half of the projected starting secondary - cornerback Vontae Davis and safety LaRon Landry - because of nagging injuries and three potentially helpful linebackers in Andrew Jackson, Josh McNary and Kelvin Sheppard.
But the most glaring problem is in the group that will be protecting Andrew Luck.
The Colts insist they're confident Holmes, who played just 12 offensive snaps last season as a rookie, can excel.
They tried to create a backup plan during the offseason by signing former Cowboys center Phil Costa in free agency. But Costa surprised Indy when he retired in April.
Thomas' injury opened the door for second-round draft pick Jack Mewhort and former Bears starter Lance Louis, who was out of the league last season, to battle for the starting job. Second-year guard Hugh Thornton has moved from the left side to the right this year, and it doesn't appear the Colts are planning to move him again.
And with backup Xavier Nixon posting on Twitter that he's about to undergo surgery for his injured leg, the Colts brought back undrafted rookie guard Marcus Hall, who was cut in June.
Holmes' injury may create the biggest obstacle. His backup is Jonotthan Harrison, an undrafted rookie from Florida, who was part of a botched snap after entering Thursday's game. The third-down miscue forced the Colts to settle for a field goal.
And in a move that may signal that Holmes could miss some time, the Colts re-signed Thomas Austin, who was released last week after playing one game with Indy in 2013.
''You know Jonotthan having to come in, I mean he's never played an NFL snap and before you know it, we hadn't planned to put him in until the second half and he's in there in the first drive,'' Pagano said. ''I think just listening, watching, I think he rose up to the challenge. Again, the snaps, we'll get that fixed.''
In a flurry of moves late Friday, the Colts also re-signed receiver Nu'Keese Richardson, who was released Tuesday, waived guard Chad Anderson and waived cornerback Brandon Burton from injured reserve after reaching an injury settlement.