America lost 2-1 at home to Tecos UAG in a deserted Azteca stadium as the arena, one of the world's most famous and imposing stadiums, was shut to the public on Sunday because of the swine flu outbreak.
It was one of three first division matches to be played behind closed doors at the weekend because of the virus which has killed 103 people in Mexico and shut down large chunks of the capital.
The games at Pumas UNAM's Olimpico Universitario stadium in the south of the capital and Pachuca, a provincial town around 100 kilometres away, were also played in empty arenas.
Pumas said they had sold all 50,000 tickets for the visit of Guadalajara, Mexico's most popular club.
The Azteca, which hosted the 1970 and 1986 World Cup finals, has a capacity of 105,000.
Carlos Adrian Morales and Mario Ortiz scored for Tecos, who had striker Fredy Bareiro sent off in the 62nd minute, and Robert de Pinho replied for the hosts.
The defeat left big-spending America fifth in Group Two of the Clausura championship and with only an outside chance of making the quarter-finals.
Pumas UNAM were held 1-1 by Guadalajara while Pachuca beat Cruz Azul 3-0 in a match played on Saturday.