The weekend's pre-season cup games marked the start of the trial, where each team is allowed a maximum of 80 interchange rotations per game.
Malthouse had already voiced his opposition last week and the new Carlton coach was even more adamant after Saturday night's 70-point belting of Fremantle at Etihad Stadium.
"It's not friendly to the players," Malthouse said of the trial rule.
"If they want medical evidence, which they've chosen to ignore, there's a very strong link to more interchanges and less injuries.
"It's not the other way around because if you fatigue players, you get fatigue-type injuries."
In his previous tenure at Collingwood, Malthouse was an architect of using the interchange bench much more frequently, especially for midfield rotations.
Speaking after his side's one-point loss to the Western Bulldogs on Friday night, Hawthorn coach Alastair Clarkson also said the 80-cap rule was too dramatic.
Clarkson said if the league wanted to cap rotations, it should consider 120 as the ceiling.
While there will be no cap during the regular season this year, there is strong speculation that the AFL will bring in some sort of restriction next season.
"There's not a person in the land who knows what's going to happen to the game when that (80) happens," Clarkson said.
"I reckon it's an enormous risk to take with what's a pretty good game we've got at the moment, just on a hunch."
While Malthouse hates the 80 cap, he and the Blues enjoyed a strong performance against the Dockers.
Eddie Betts was best afield as Carlton won 0.18.16 (124) to 1.6.9 (54).
On Friday night, the Bulldogs defied predictions of gloom this season when they beat Hawthorn, the early premiership favourites, 1.10.6 (75) to 2.8.8 (74).
In Wangaratta on Saturday, Ricky Petterd's nine-point goal with three minutes left put Richmond in front for the first time in the second half and they beat Essendon 2.8.20 (86) to 1.10. 9 (78).
Geelong dominated at home on Saturday, thrashing Adelaide 2.16.12 (126) to 1.8.6 (63).
Gold Coast fought back in the final term at Townsville, but North Melbourne held on to win on Saturday 2.13.6 (102) to 1.11.14 (89).
Brisbane's Aaron Cornelius starred with five goals in Wagga Wagga as they held on to beat GWS by three points, 1.12.19 (100) to 2.12.7 (97).