Federer had cruised through the tournament so far, winning four matches in straight sets to confirm himself as a heavy favourite to retain his Melbourne crown.
However, No 19 seed Berdych immediately challenged him in the early stages of their match, breaking him in his opening service game and jumping out to a 3-0 lead.
Federer, by contrast, appeared flat and could not find the rhythm on his first serve that had been so apparently easy for him in the first week.
Berdych served for the first set with half an hour gone and Federer still below par, but the Swiss managed to force his first two break points of the match.
At 15-40, Berdych responded with a deep serve into the body which Federer could not properly return and the Swiss challenged the line-judge’s call.
The Hawkeye system left the crowd and players waiting until umpire Fergus Murphy announced that while the graphic could not be displayed, the call had been the right one and the ball had caught the line.
Federer, having challenged incorrectly, was then told he had lost one of his challenges, a decision with which he seemed to take issue.
Jeers and whistles from the crowd rendered much of the conversation indecipherable but Federer continued talking to the chair for up to a minute.
“Are you comfortable with this? It’s your call,” he seemed to say repeatedly.
Federer lost the next two points but went onto save a set point in the game and did eventually break back.
The world No 2 forced a first-set tie-break which he then dominated to take it 7-1 and take the lead.