Harper batted second in Washington's lineup against the Cardinals.
In the sixth inning on Saturday, Harper hit a comebacker to the mound and was easily thrown out at first base. The two-time All-Star was replaced in left field by Kevin Frandsen the following inning.
Following the eventual 4-3 loss, Nationals first-year manager Matt Williams cited "lack of hustle," for removing Harper. "That's why he came out of the game," Williams said. "He and I made an agreement, this team made an agreement, that when we play the game, that we hustle at all times."
On Saturday, Harper said after the game: "I respect what he did. That's part of the game."
In the ninth inning with Washington trailing 4-2, Frandsen drove in a run on a groundout, but the inning ended one batter later.
The program from Saturday night's Nationals home game featured a photo of Harper and the slogan "Nothing but Hustle."
"Yes, that is the cover of the Inside Pitch magazine, the game program handed out at home games," a Nationals spokesman told ESPN's Darren Rovell. " 'The "Nothing but ...' slogan is part of our season-long advertising campaign that plays off 'Nothing but Natitude.' Many of the players have been assigned a specific word as part of the campaign. For example, Jordan Zimmermann's is 'Nothing but command.' I believe Ryan Zimmerman is 'Nothing but clutch.' Harper's word, obviously, was hustle."
The manager's decision and postgame comments also served as a reminder to others in the clubhouse about their on-field conduct.
"You expect to play hard every day. That's what we do as baseball players. That's what we do as Nationals,'' right-hander Tanner Roark said before Sunday's game. "Matt expects that and we expect that out of ourselves. You learn from it and move on.''
In his third season, Harper is batting .295 with one home run in 16 games this season. He previously missed Wednesday's game in Miami because of tightness in his quadriceps.
The Associated Press contributed to this report.