Naturally it depends on what you are doing, but for regular Win16/Win32 code that uses Windows type macros and no low level tricks pretty much so.

And even if you take Win16 out of the picture, Win32 exists since 1995.

I'm not doubting that the windows API has remained stable. I'd like to try if the tooling (e.g. IDE config format or makefile equivalent) has also received the same amount of attention to backward compat

Again, depends on what you were using.

Borland C++ or Watcom, probably not much help.

Delphi, C++ Builder, Visual C++ for Windows 95/2000 mostly work, naturally you might need to use the IDE wizards to upgrade project files, or fix tooling paths on the makefiles.

https://github.com/dbremner/cake is a 16-bit Windows 1.0 application that I ported to Win64; most of the changes weren't required.