The Men's Olympic team were facing the Clever Boys in a preparatory friendly on Monday morning, ahead of the Summer Games which kick off on August 4.
Khune came on as a substitute for Jody February at half-time while Owen Da Gama's men were trailing 1-0.
However, the Kaizer Chiefs shot-stopper limped off the pitch less than 10 minutes later, with what seemed to be an injured left ankle, and was replaced by reserve keeper Nkosingiphile Gumede.
It is yet to be determined how long the 29-year-old will be out for.
Chiefs recently announced that Khune will play in the Carling Black Label Champion Cup against Orlando Pirates on July 30 despite the Olympic opener in Brazil just five days later.