The trio were joined by Elinor Barker as they won the 4,000m women's team pursuit in 4:32:721 - nearly 40 seconds quicker than their nearest rivals in Manchester.
King, Rowsell and Trott won London 2012 gold over 3,000m and claim the change in distance and addition of a new team member - to bring the event parallel with the men's programme - is a challenge to savour.
It will be great to see how we progress this year," said Rowsell.
"We've got the three kilometre world record, which should stand forever, and it's exciting to be able to try and do that all over again with the new event.
And King insists the team - and their rivals - will only get quicker as they get used to the new discipline.
"It's quite different really, an extra four laps, but there's an extra rider as well so that changes things. We're learning all the time and think we can only go quicker," she said.
"We took a similar approach to when we were a three, it's all about maintaining speed."
Jess Varnish claimed her fourth national title as she added to victories in the sprint, team sprint and 500m time trial with a win in the women's keirin.
Treble Olympic gold medallist Jason Kenny teamed up with Matt Crampton and Kian Emadi to win the men's team sprint while Trott and King also won the women's madison.
I just wanted to get a good week's training," said Varnish. "I've never actually won a keirin before, and I'm quite surprised that I actually have."
Meanwhile, Tour de France winner Chris Froome admitted there was no excuse for a poor British showing in the men's road race at the World Championships in Florence.
Only 48 of the 208 starters completed the 272.5km course - and not a single British rider was in that number.
Bad weather made conditions treacherous and Froome, who was leading a team that included Sir Bradley Wiggins, Mark Cavendish, Luke Rowe, Steve Cummings, Josh Edmondson, Ian Stannard and Geraint Thomas, pulled out with 80km remaining.
"The conditions are the same for everyone. We've got no excuse, we just weren't there," he said.
"It's a big disappointment, especially having made it such a big objective but with these conditions it just wasn't meant to be.
"The first three laps on the circuit there were crashes everywhere. The weather hasn't let up an it's rained solidly all day.
"Given we've come up empty handed we'll have to go back to the drawing board [ahead of the Rio Olympics]."
Double Olympic champion Geraint Thomas described the race as carnage with Portugal's Rui Costa eventually taking the rainbow jersey, beating Spain's Joaquim Rodriguez in a two-man sprint.