brundlefly
It occurs to me that another source of variation from one recording of the same source to the next will be the performance of the analog pre-amps and anti-aliasing filters on the inputs. It's doubtful they will behave exactly the same way every time. That's a lot of what your serial re-recording test is going to be measuring.

Ah - it may be this.  I had not considered anti-aliasing filtering either, and it might account for a small loss.  However, I think after watching the video linked above that I made a mistake in how I set CueMix to get unity gain, so I have an offset.  I'm going to try it all again. 

thedukewestern
https://www.youtube.com/watch?v=-8_b1xfOrJY    heres a video I made that deals with examining the outside world with the inside world.. signal levels on your interface preamps etc.

I am using line inputs and I'm not using the two liquid channels but rather another line pair.  There is a gain control that may not be allowing me to match things exactly.  Anyway, let me see what the video yields, thanks for the link.
 
 

An individual re-recording may not be anywhere near unity gain, depending on how well calibrated the the analog output level and input sensitivity of the interface is, and whether they are both calibrated to -10dBu or +4dBV line level (not necessarily the case). There should be controls on at least one side (output or input) to allow you to match them.
 
At that point, you should get relatively repeatable recordings of a fixed source but you'll still have small, random variations in peak dB readings due to the inconsistency of the analog circuitry and the relative timing of the sampling, above and beyond the generational loss of 'fidelity' you're wanting to investigate.
 

thedukewestern
https://www.youtube.com/watch?v=-8_b1xfOrJY    heres a video I made that deals with examining the outside world with the inside world.. signal levels on your interface preamps etc.

Life got in the way for a while there.  **THIS**  you nailed it, for me.  I followed your video and while I found it touchy to get exact levels (things were jumping around with the tiniest movement of the gain knob on my interface) but was able to get things matched up pretty well.  Thanks, this was not intuitive for me - I still find the Focusrite Mix Control interface hard to grasp.