"Without any gray areas, [I] told them exactly what was going to happen," Maddon said of the meeting. "And it's happening."
Upton gave a lackadaisical jog to first base in his final at-bat, after hitting the ball back to pitcher Edward Mujica, and Maddon would have preferred to remove the outfielder immediately but was out of position players.
The 23-year-old Upton has often shown flashes of superior effort, including a stellar catch in the seventh inning on Tuesday, retiring Cleveland's Ben Francisco on a deep fly ball.
"You just can't pick and choose when you put your effort out there," Maddon said. "It has be all the time."
Typically a laid-back manager, Maddon said he addressed the matter directly following Tuesday's game and that Upton "listened" accordingly.
"It just comes down to respect," Maddon said. "Respect for yourself and respect for the organization."
Following Wednesday's game, Upton took no issue with Maddon's stance.
"I didn't run it out," Upton admitted. "Lesson learned. It's over. Done with. Moved on."
Maddon was also ready to put the situation behind the club.
"It's a dead issue," he said. "I want to be consistent in what I say. He will be back [Thursday]."
In Upton's place, the Rays gave recently recalled infielder Ben Zobrist a start in center field on Wednesday and inserted Willy Aybar into the shortstop role for Zobrist.
Although the club is still without regular shortstop Jason Bartlett in the field, the infielder was in Wednesday's lineup as the designated hitter.
During Sunday's game, Bartlett was hit by a pitch on his right index finger, and although he has taken batting practice, the shortstop sat on both Monday and Tuesday.
"He's OK to hit, he just couldn't throw today," Maddon said.
Bartlett was seen playing light catch prior to Wednesday's game and the Rays are hopeful he will be ready to join the defense for Thursday's game in Seattle.
"It's soon," Maddon said of Bartlett's return to shortstop.
Backstop Shawn Riggans -- who was also hit by a pitch in Sunday's game -- was out of Wednesday's lineup as well.
After taking a blistering fastball in the chest, Riggans reported to be more sore on Tuesday than he was initially and the club decided to use caution with its backup catcher.