I now know what the problem trigger is. It's my Steinberg interface (or, more accurately, the driver).

I hooked up my NI Komplete Audio 6, and instrument low latency mode works perfectly. With the project open, I flip back to the Steinberg interface, and no instrument sound. 100% reproduceable.

I'm using an MR816X, which is Firewire. The only other report I've seen so far was with a Steinberg USB interface.

Please put your specs in your signature. See mine for a link.

Does this happen with a new empty song with that MIDI interface?

It's the audio interface I'm changing, not the midi. All I had hooked up for midi at the time was my Novation SL61 via USB.

I tried a new song, and the results were strange.

* Rebooted, started S1 fresh, created new song
* Added one instrument track, added Mojito , played fine with dropout protection (DP) on
* Added some audio tracks (dragged them in from old Cubase project, didn't record them), DP on OK
* Added some plugins, DP on OK
* At this point, I think it's OK, and it's related to a pre 3.5 song only. So, my "testing" is over.
* Have some ideas, start to mess around with that same song, not particularly structured. At some point, I go to the instrument, and it doesn't work with DP on
* I bring up the undo history. If I undo all the way back to where I first added the audio tracks, the instrument plays with DP on. Redo adding the tracks, it stops.

BTW, I did add my system specs to my sig, but it's not displaying. I had support manually change my handle to j0001s instead of showing my real name. Since then, some forum features don't work. I guess that the sig is one of them. I've added the info myself below.

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Win 7 SP1 64 bit (all updates), I7 4 core, 8 Gb, Dual monitors (1920x1200, 1920x1280), Steinberg MR816X, 3xUAD Solo, TC Electronic Powercore Element, Novation SL61, Faderport, Studio 1 Pro 3.5, Cubase 8.5

At some point, I go to the instrument, and it doesn't work with DP on.

What instrument have you been talking about all along?
Virtual or physical. Name?

All virtual instruments. Tried a ton. The test case above used Mojito.

OK well we're talking you should open a support ticket anyway

Will do later tonight.

Ok this needs to be consolidated to see the symptoms and debug path.

Setup
S 3.5, Windows 7,Yamaha Steinberg FW interface (MR816x), Novation SL61

Condition:

• No issue with audio recording in low latency mode.
• Midi is showing as being received on the instrument track. There is either no sound, or sometimes (haven't pinpointed the conditions), the instrument will spit out the notes more than 5 seconds after I've played them. <== SYMPTOM 1
• The problem trigger is. It's my Steinberg interface (or, more accurately, the driver). MR816X, which is Firewire Swap out all works OK, I flip back to the Steinberg interface, and no instrument sound. 100% reproducible. <== SYMPTOM 2

Test:
1. Rebooted, started S1 fresh, created new song
2. Added one instrument track, added Mojito , played fine with dropout protection (DP) on <= SYMPTOM CHANGE 1
3. Added some audio tracks (dragged them in from old Cubase project, didn't record them), DP on OK
4. Added some plugins, DP on OK
5. At this point, I think it's OK, and it's related to a pre 3.5 song only. So, my "testing" is over.
6. Have some ideas, start to mess around with that same song, not particularly structured. At some point, I go to the instrument, and it doesn't work with DP on <== SYMPTOM 1
7. I bring up the undo history. If I undo all the way back to where I first added the audio tracks, the instrument plays with DP on. <== SYMPTOM 3
8. Redo adding the tracks, it stops.

FYI there is a pattern slowly emerging with older OS versions.
1) Start a new test song repeat 1-6 without adding plugins.
2) Open your performance monitor and watch instrument latency closely.
3) Once it is working step 4-5 save sn and reboot S1 to continue testing.
Let us know the findings.
Thanks