Funny you should ask. I spent a while a few years ago messing around with csound, synthesizing Bach chorales in a number of tuning systems. You could tell a difference, IIRC, even with kind-of-unpleasant synthesized tones. I'd have to go back and look up what I did, but I must have done both equal temperament and (some kind of) just intonation.
You can absolutely tell a difference! Probably more so with synthesized sounds, which don't have natural pitch deviations or complex harmonics. The beating you hear in any even-tempered dyad other than an octave is really obvious with sine or square waves, presumably especially so with the sustained notes in a chorale :)
There's a blues guy Willie McBride who has recorded blues in just intonation. It's kind of interesting to listen to, not for everybody, but if you're OK with indian and other non-western-classic music, it's worth checking out.
