See also for the opposite: Modifying a virtual file .
For completeness, I will send back the instructions of my answer. In the end, this question can be combined with another.
Since --no-site-packages is the default behavior in recent versions ( > = 1.7 ), you just need to recreate virtualenv with the command:
$ virtualenv $PATH_TO_VIRTUALENV
or in older versions where --system-site-packages was by default:
$ virtualenv --no-site-packages $PATH_TO_VIRTUALENV
replacing $PATH_TO_VIRTUALENV with the path to the existing virtualenv. This will reinstall virtualenv, reconfigure it to prevent access to global packages, keeping the packages already installed there.
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