The stand-in boss made sweeping changes after the Blues beat Liverpool 2-1 in Saturday's FA Cup final and they were picked apart by the rampant home side who gained some revenge for their Wembley disappointment.
Chelsea must now win the Champions League final against Bayern Munich to play in Europe's elite club competition next season following the defeat at Anfield.
With one match remaining, Chelsea are five points adrift of fourth-placed Tottenham Hotspur and four behind fifth-placed Newcastle United, both of whom are in a final-day scrap with Arsenal to determine the Champions League qualifiers.
It is the first time Chelsea will have finished outside the top four since the 2001-02 season.
Yet Di Matteo said he had no regrets, despite his team beginning the night still in contention for a top-four berth.
"I have no regrets, the effort was there but we came up against an inspired Liverpool side," Di Matteo, who left out the likes of Frank Lampard, Didier Drogba, Ashley Cole and Juan Mata, said.
"We have had some very intense games, the last one three days ago. It's hard to keep getting yourself going every three days. Since I've been in charge we've played every three days and it's been a big ask and a big demand.
"I just thought with fresh legs and new energy we could have had a better chance tonight."
Liverpool manager Kenny Dalglish, whose side have managed just six league victories at Anfield from 19 matches, said: "That performance was on a par with many this season but we've not had the results those deserve."
Liverpool's win saw them leapfrog Fulham into eighth place on goal difference and they can still finish above Everton if their result at Swansea City on Sunday is better than that achieved by their city rivals at home to Newcastle United.
"We've got a bit of silverware back in the cupboard after six years, we're back in Europe at the first time of asking, we were near to winning the FA Cup final. Not a bad first season," Dalglish added.
"They (the players) understand better than everyone (the club's expectations) and they're probably more educated than most of those people (critics).
"I think the players will be happy to finish on a high at Anfield. That performance was on a par with a lot of other performances where we've not had the result. Tonight we got that.
"For us it was a satisfying evening. Hopefully the supporters go away feeling very happy. Hopefully the boys have put a wee smile on their faces."