It was reported on Friday evening that the Spaniard stormed out of a meeting and informed the players that he did not want to manage them.
Now, the Championship club has confirmed that the 42-year-old did not take training this morning and will not be in the dugout for tomorrow's league game at The Valley.
A statement from Boro read: "Middlesbrough Football Club can confirm that Aitor Karanka did not take training on Saturday morning. Assistant head coach Steve Agnew will take charge of the team for Sunday's fixture at Charlton Athletic.
"We appreciate supporters will want to know further details, but at this time the club cannot make any further comment and is solely focused on Sunday's game at The Valley."
Middlesbrough are currently second in the Champions League table in an automatic promotion spot, four points adrift of leaders Burnley.
Karanka, who joined Boro in November 2013, has won 67 of his 128 matches in charge.