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We have ~10 users who run legacy (16-bit DOS on windows 98) data entry software against a Windows 2003 file server. Files are accessed directly (i.e. it runs as if it's a client application with network files constantly being accessed, not as a client/server app).

Lately, the performance of this application has been atrocious. 15 seconds to run a tiny report, or open up a new screen. But when we look at the server's performance metrics, there doesn't appear to be any problem. Low IOPS, no average disk wait, low read/write bytes, almost 0% CPU usage, tons of free RAM, etc. We've looked through every metric and seen nothing that seems even remotely close to the limits of the server.

We are in the process of replacing the software, but need to make it work for another year until our transition is complete. Any ideas on how we can determine the source of the issues?

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It might be some network protocol problem. Did you take any traces? If not, you should - fire up Wireshark, run the "tiny report" and evaluate the data. – the-wabbit Nov 22 '11 at 23:19
syneticon-dj. This must have been it ... we didn't see any issues in wireshark, but when we moved one user to a different switch everything cleared up. No idea why. – Beep beep Dec 4 '11 at 14:04
could be anything on the switch ;) – TomTom Dec 4 '11 at 14:24

Per my comment, it turned out that this was a network issue. No idea why, but moving one user to a different switch fixed it.

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Process monitor from Sysinternals (Now part of Microsoft) will give you all the info you need:

Simply run it, prior to running your app, make sure you set up the filters and options correctly (It produces a huge amount of info!) then sit back, while your app is run and observe what shows up in the monitor while the slow down takes place.

Given that you can reproduce your scenario, you should after a few runs be able to see what's causing you the problems.

There are other tools on the Sysinternals site that are likely to help you too.

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