Oviedo and Espanyol players refused to move for the first 15 seconds of their match on Friday as the teams staged a protest at La Liga's decision to host a game in Miami.
In August, the Spanish football federation (RFEF) approved plans to move Villarreal's fixture against Barcelona to the Hard Rock Stadium on 20 December - a decision which has sparked discontent in Spain.
The Spanish Footballers' Association (AFE) said in a statement they had coordinated with the captains of each La Liga club to "protest symbolically" against the league's "lack of transparency, dialogue and coherence".
La Liga refuted the accusations, external earlier this week, stating a "willingness to meet" with the AFE to "foster useful dialogue that allows explaining the project".
The protest by Oviedo and Espanyol players was not shown on live television as cameras cut away to the exterior of the stadium.
The AFE's statement added that replica demonstrations will follow at the remaining La Liga games this weekend.
The union has not asked Barcelona and Villarreal players to participate, however, to avoid it being "interpreted as a possible measure against any club".
The statement continued: "In the face of La Liga's constant refusals and unrealistic proposals, the Spanish Footballers' Association categorically rejects a project that does not have the approval of the main players in our sport and demands that the employers' association create a negotiating table in which all information is shared and the exceptional characteristics of the project are analysed, the needs and concerns of the footballers are addressed, and the protection of their labour rights and compliance with current regulations are guaranteed."
The BBC has contacted La Liga for comment.
Confirmation of the Miami game followed a decision by the Italian football federation (FIGC) to sanction a Serie A match between AC Milan and Como to be played in Perth, Australia in February.
Earlier on Friday, Barcelona coach Hansi Flick said his players were not pleased at having to travel to the US to play a La Liga game.
"My players are not happy, I am not happy, but La Liga decided that we will play this game," Flick said at a news conference.
Real Madrid expressed opposition when the fixture was announced, stating the consequences would be "so serious", while Uefa "reluctantly" approved the move.
This week, RFEF president Rafael Louzan said the move is "good for football", while Barcelona president Joan Laporta praised the decision when it was confirmed, adding the match will "definitely be a great show".
