Khune, who is due to play in the Carling Black Label Champion Cup on July 30, limped off the field 10 minutes after replacing fellow goalkeeper Jody February at half-time at Milpark Stadium.
The Kaizer Chiefs man seemingly hurt his left ankle, prompting the introduction of Nkosingiphile Gumede. It is unclear how long Khune will be out for.
Wits winger Vincent Pule’s first-half goal was cancelled out by Andile Fikizolo in the second, as both sides created minimal opportunities in front of goal.
Gavin Hunt fielded a full-strength side to test the Olympians at the Bidvest Stadium offering coaches Owen Da Gama and Thabo Senong a decent view of their state of readiness ahead of the Summer Games, which kick-off in less than three weeks.
Da Gama fielded an experimental side in the first half, as Deolin Mekoa featured at left-back and Kwanda Mngonyama turned out in the centre of the park to allow Rivaldo Coetzee and Eric Mathoho to partner in central defence.
Goalkeeper Jody February had a quite opening 15 minutes as Wits looked to build up some rhythm in their build-up play.
He was then left exposed by his defence when experienced winger Elias Pelembe broke away on the counter-attack, catching Mekoa out on the blind-side, and delivered a low cross to find an unmarked Pule, who tapped home at the back-post.
New Wits signing Eleazar Rodgers also forced February into some fine diving saves, but was unable to get on the scoresheet before being substituted at half-time.
At the start of the second half, Da Gama introduced Itumeleng Khune in place of his Ajax Cape Town counterpart, while Brazilian-based Tyroane Sandows came in for Chippa United-bound Menzi Masuku, as Wits fielded a completely different 11.
Just over five minutes after the restart, Khune was involved in a collision with an unnamed Wits striker which prompted reserve goalkeeper Nkosigiphile Gumede to come on in his place.
Midway through the second stanza, Da Gama introduced new Orlando Pirates striker Thabiso Khutumela and Golden Arrows winger Andile Fikizolo, which later yielded an equaliser after the former beat the Wits off-side trap to pick out the latter, who did well to beat Moeneeb Josephs.
Gremio midfielder Sandows also forced a great save from Josephs a few minutes later when his dipping free-kick looked set to sneak in under the cross-bar.
The visitors created a few more opportunities towards the end of the match but were unable to get the beating of the former Bafana Bafana international, who ensured the score stayed at 1-1.
Wits: Peters (Josephs), Hlanti (Sheppard), Mkhwanazi (Lecki), Hlatshwayo (Khumalo), Nhlapo (Zulu), Monare (Motshwari), Wome, Phiri Jr (Shongwe), Pule (Klate), Pelembe (Mosiatlhaga), Rodgers
SA Under-23: February (Khune) (Gumede); Mekoa (Moerane), Coetzee, Mathoho, Mobara, Mngonyama (Fikizolo), Mvala (Molepe), Modiba (Morena), Masuku (Sandows), Motupa, Morris (Kutumela)