Olympic 400 metre champion Mack Horton will not be defending his title in Tokyo after failing to qualify at the Australian trials Saturday, stunned by Elijah Winnington who swam the world's fastest time this year.

Horton, who beat drug-tainted Chinese star Sun Yang for the title in Rio then refused to stand on the podium with him at the 2019 world championships, has been struggling for form this season.

The 25-year-old needed to come good on day one of the trials in Adelaide and he gave it his all, but his 3mins 43.92secs wasn't enough despite being one of the quickest globally in the last 12 months.

He was narrowly in front at 300m but Winnington came storming back to win in a sizzling 3:42.65 with Jack McLoughlin (3:43.27) pipping Horton for second with only the top two making the grade.

"It's really incredible," said 21-year-old Winnington. "I've changed coaches in the past year and it's been incredible for me. We've done so much work and that's why I had full confidence in my last 100."

Despite missing out on the 400m, Horton could still make the plane to Tokyo in the 200m on Sunday.

Swimming Australia has set the bar high at the six-day trials -- to qualify, athletes must match or better the time needed to make the final at the 2019 world championships.

A maximum of 56 swimmers -- 28 men and 28 women -- can be selected in a sport that has traditionally been the medal-winning backbone for Australia at the Olympics.

Emma McKeon, part of Australia's gold-medal-winning 4x100m relay team at the 2016 Rio Olympics, was also on fire, clocking the world's second fastest time of the year in the 100m butterfly to easily qualify.

The 27-year-old made the turn under world record pace before touching in 55.93, behind only China's Zhang Yufei (55.73) this season as she zeroes in on Swedish great's Sarah Sjostrom's 2016 world best of 53.48.

"I haven't done a PB in that event since 2017 so it's been a while and I felt like it was time," she said after setting a new Commonwealth and Australian record.

"It's not relief, just joy, It's been a long time coming for everyone," she added of a difficult Covid-hit year.

Brianna Throssell was second in 57.11, fractionally outside the qualifying time.

McKeon, who won four medals at Rio, is shaping up as one of Australia's best hopes in Tokyo. She will also race the 50m, 100m and 200m free in Adelaide.

Brendon Smith was another to smash an Australian record in the 400m individual medley, hitting the wall in 4:10.04 to comfortably qualify in the world's fourth fastest time this season.

Lee See-Bom swam a new personal best to finish second and also make the Tokyo team.

"I've been putting in a pretty good block of training recently. I knew it (the record) was on the cards but I needed a very good race to do it," said Smith.

In an upset, neither the fancied Zac Stubblety-Cook nor Matt Wilson posted fastest enough times in the 100m breaststroke.

Stubblety-Cook touched first in 59.69, but outside the 59.21 needed, with former 200m breaststroke world record holder Wilson in fourth.

Wilson will attempt to make the grade in the 200m on Tuesday.