Spoelstra ruled all three out for Tuesday night's game, with James battling hamstring tightness and Wade and Chalmers dealing with right ankle sprains.
"Everybody's human," Spoelstra said.
It's the second straight game in which Miami will miss those three starters. The Heat won in San Antonio 88-86 on Sunday without them. Spoelstra said all three players are improving, and none of them were available for comment after Miami's shootaround Tuesday morning.
"We're not panicked about it," Spoelstra said. "We're being very proactive and aggressive with the treatment and rehab, just to make sure we can contain their injuries where they are. You get into 70-plus games, where we are right now, we feel very fortunate. These are minor nicks and knacks that happen over the course of a long season.
"These are minor things right now," Spoelstra added. "Minor things can turn into much bigger things if they're not addressed."
Meanwhile, while the Heat were dealing with roster subtractions, the Knicks got a roster addition. Center Tyson Chandler was back in the starting lineup after missing 10 games with a back problem.
"Every game is important for us no matter who plays," Knicks coach Mike Woodson said.
Chandler was replacing Kenyon Martin. Woodson left the remainder of the Knicks' starting five intact, with Chandler joining Carmelo Anthony, Raymond Felton, Pablo Prigioni and Iman Shumpert, who was back in Miami for the first time since enduring a severe knee injury in the opening round of last season's playoffs.
Miami has already wrapped up the No. 1 seed in the Eastern Conference playoffs. Miami's magic number for clinching home-court advantage throughout the NBA Finals was five -- any combination of Heat victories or Spurs losses.
Wade took a misstep last Wednesday in the Chicago game, the one that snapped Miami's 27-game winning streak, and tweaked his ankle. Chalmers also injured his ankle in that game, and the Heat said James' hamstring tightness started early in Miami's win at New Orleans on Friday. James finished that game with 36 points, sitting out the fourth quarter.
Woodson said he had no doubt that the Heat injury concerns were legitimate.
"They go into San Antonio and they win without those three guys, so I say they're a pretty good basketball team," Woodson said.
The Heat played 18 games in March alone and have won 29 of 30 entering the matchup with the Knicks, who took two of the first three meetings between the clubs this season.
The Knicks enter Tuesday night with the NBA's longest current winning streak, eight straight games.
"They're fighting for the 2 seed with Indiana, so this is a big game for them," Heat forward Shane Battier said.
After the Spurs game, Battier called the roster that the Heat used in San Antonio "the fighting clowns."
He had a new moniker Tuesday.
"The Expendables' will be ready to go," Battier said.