He was taken off with 25 minutes of Sunday's game still to go and with the outcome anything but settled as Juventus led Hellas Verona 2-1, which turned out to be the final score.
Having been substituted only once during the whole of last season's triumphant campaign, another early exit on Wednesday could indicate that, at 34, Pirlo might be starting to lose his untouchable status.
Pirlo, whose beard gives him a startling resemblance to American actor Chuck Norris, has enjoyed a second wind since moving to Juventus two years ago from AC Milan, masterminding two successive Serie A title-winning campaigns.
During that time, he has pulled all the strings in midfield, nonchalantly spraying passes around the field and dictating the rhythm of the team from his favoured position just in front of the Juventus defence.
He has also played a part in Italy's revival under Cesare Prandelli and in June received the ultimate accolade when he had his name chanted by the fickle and notoriously hard-to-impress Maracana crowd during the Confederations Cup in Brazil.
But now there are signs that, with Juventus again battling on two fronts in Serie A and the Champions League, Pirlo might find himself being rotated like everyone else.
Juventus have plenty of alternatives in midfield with Claudio Marchisio back from injury, Frenchman Paul Pogba rapidly improving and irrepressible Chilean Arturo Vidal.
There have even been instances of Pirlo getting caught in possession and giving the ball away.
Pirlo's reactions will also be under scrutiny after he went straight down the player's tunnel after his substitution on Sunday, earning a black mark from Conte.
"I will tell Andrea that when he leaves the field after being substituted, he must remain on the bench and wait for his team mates," Conte said after the game. "The good thing is that he has rested for 25 minutes and will be ready for Verona."
Juventus, one of five unbeaten teams after four games of the new season, are level on 10 points with Fiorentina and Inter Milan and two behind leading pair AS Roma and Napoli.
Rafael Benitez's Napoli should make it five wins out of five when they host Serie A debutants Sassuolo, who have yet to collect a point and were thrashed 7-0 at home by Inter on Sunday. Roma, also with a perfect record from four games, visit Sampdoria, who have two points.
Inter, a different team from the one which finished ninth last term, host Fiorentina in the most attractive of Wednesday's games.
"The players seem to be proud wearing this shirt. When you win you feel you're part of an ideal," said club president Massimo Moratti. "We've spent intelligently, bringing in lots of youngsters who have come in for criticism but are showing how talented they are."