The German mentor said while Khune is recovering well from his shoulder, the 31-year-old shot-stopper hasn't even started doing ball work with goalkeeper coach Lee Baxter.
"Regarding Itu Khune, what are you expecting me to answer? Is he fit? Let's be honest, seriously, it's nearly the end of April. Not even now, not even over shoulder. There's no ball involved at all," Middendorp told reporters at the FNB Stadium on Tuesday.
Asked if Khune stands a chance of featuring in Amakhosi's remaining four games of the season, Middendorp said: "There's no chance. 100 percent [no chance].
He went further to say people expecting Khune to be included in Bafana Bafana's final squad for the Afcon tournament between June and July aren't serious and honest to themselves.
"If somebody dreams about having Itu Khune into Afcon when he's not even at training he's not serious and [he would] dishonest to do something like that," said Middendorp.
Middendorp's statements would be seen as a jibe at Stuart Baxter, who previously said Khune's possible inclusion into the Bafana squad is firmly on the Amakhosi technical team.
"That's my opinion, but if the national team coach [Baxter] has an idea to call Khune for Afcon, it's his decision. Our medical department definitely has other ideas," added Middendorp.
Furthermore, the 60-year-old tactician warned Khune that he would have to work twice as hard to regain his No.1 jersey at the club next season.
With Daniel Akpeyi and Bruce Bvuma slowly finding their feet, Middendorp believes Khune will face stiff competition from his two colleagues.
"Secondly, he should make it really happen to concentrate and have a very clear structure to prepare himself to have a good start to the [next] season. We have Bruce and Daniel, who's more and more settling in. We have goalkeepers who have high competitive quality level," concluded Middendorp.
The news of Khune will come as a massive blow for the South Africans, who would have hoped to see him shine at the Afcon tournament which will take place between June and July in Egypt.
Bafana Bafana have been drawn in Group E alongside Namibia, Cote d'Ivoire and Morocco.