Khawaja wasn't eligible to open in Australia's first innings at a sold-out Perth Stadium on Friday after spending too long off the field during England's knock, dealing with a tight back and then back spasms.
It meant Marnus Labuschagne had to open alongside debutant Jake Weatherald, and Steve Smith was forced to come in at No.3.
Weatherald fell for a second-ball duck, Labuschagne struggled to nine off 41 balls while Smith copped a series of body blows on the way to 17.
Khawaja made just two batting at No.4, meaning he has now failed to post a half-century in his past 11 Test innings.
Australia ended the day at a disastrous 9-123 in reply to England's 172.
A sore-looking Khawaja trudged off the field again on Saturday to receive more treatment with England struggling at 6-104 in their second dig.
The 38-year-old had dropped a catch at slip earlier in the innings, with the missed chance placing further pressure on Khawaja to keep his spot ahead of the likes of Mitch Marsh and Josh Inglis.
Khawaja played golf with Mitchell Starc on Thursday, just 24 hours before day one of the Ashes.
Fans and experts have been quick to criticise the left-hander for that decision, but CA chief executive Todd Greenberg didn't have an issue with it.
"It (playing golf before a Test) has held him in good stead over the last couple of years. It's not uncommon for a lot of them to play golf a day prior," Greenberg told SEN.
"Did that correlate to any of the issues? I personally don't think so.
"Usman's a very seasoned campaigner, he knows his body well, and he knows how to get himself prepared for cricket. So I think that (saying golf was to blame) is drawing a long bow.
"A lot of people talk about cricket being played between the ears.
"So getting some time away from being in your hotel or at training I think is really important.
"I noticed Mitch Starc played golf with him, so it certainly didn't impact his performance (given he snared 7-58)."
Starc also defended Khawaja's preparation.
"It's interesting that there are lots of opinions from people that aren't in the change room," Starc said.
"We'll let the opinions be the opinions.
"We know what's going on in the change room. It was unfortunate that was the case - he had a back spasm, there's not much you can do about that."
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