Typically on 486 motherboards with direct-mapped cache, if your L2 cache is in write-back mode, the board will cache up to 32 MB of RAM. If it the L2 cache is in write-through mode, it will cache up to 64 MB of RAM.
If you are using DOS with 64 MB of RAM and L2 in write-back mode, you likely will not notice any performance penalty if your application uses less than 32 MB of RAM. DOS uses RAM from the bottom up. Apparently, Windows uses RAM from the top down, so for the same situation, the uncached RAM gets used first and performance will be as if you don't have any cache installed, that is, until the system uses up 32 MB of uncached RAM and starts to use the cached RAM.
So your question as to how much performance loss is that of how much loss is there without any cache for certain situations. Off the top of my head, I think the performance loss is in the ballpark of 15%.
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