Middlebrooks was not in the lineup for Monday's game against the Oakland Athletics, and no timetable has been set for his return.
Boston's rookie slugger experienced tightness in his left hamstring after stealing second base in the ninth inning of Sunday's game against the Seattle Mariners.
"He feels better today than he did yesterday, and better than he expected to be today," Red Sox manager Bobby Valentine said. "It's not discolored, and that's good, but it's going to be some days."
Middlebrooks dealt with hamstring issues in May, and this recurrence could lead to a trip to the DL.
"We won't let the calendar dictate it," Valentine said. "We'll just see how he feels. We're not sure. Maybe DL, maybe a few days. It's just one of those injuries."
Nick Punto is starting Monday at third base and batting ninth. He's batting just .200 this season with one home run and seven RBIs in 78 at-bats.
Valentine was non-committal as to whether Punto would see the bulk of the playing time at third while Middlebrooks is out.
Lillibridge primarily has played first base and outfield this year, and both of his starts with the Red Sox since the June 24 trade have come in center field.
"I've seen him take ground balls (at third base) twice. He could play there," Valentine said. "Let's put it this way, if something happens to Nick tonight, we'll see him there."
Middlebrooks' injury leaves the Red Sox with Lillibridge and catcher Kelly Shoppach as the only position players available off the bench. Valentine anticipates a roster move could be coming.
"We're trying to evaluate all of the possibilities," he said. "(General manager) Ben (Cherington's) been so good about getting the right people here at the right time. I'm confident he'll be able to do it again."
With 13 pitchers on the roster, the Red Sox could send one down to add some position player depth.
Saturday's doubleheader against the New York Yankees complicates the situation, although the Red Sox will be allowed to add a 26th player to the roster for the two games under a new rule allowing for an expansion of rosters for twinbills.
Jimmy Durkin is a contributor to ESPNBoston.com.