Which of the following is the most effective way to protect an application server running...

Air-gapping is the most effective way to protect an application server running unsupported software from network threats. By physically isolating the server from any network connection (no wired or wireless communication), it is protected from external cyber threats. While other options like port security or a screened subnet can provide some level of protection, an air gap offers the highest level of security by preventing any network-based attacks entirely.

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CompTIA Security+ SY0-701 Course Content: Domain 03 Security Architecture​.

CompTIA Security+ SY0-601 Study Guide: Chapter on Secure System Design​.