Low is "off" or connected to common or ground, whereas high is on. Therefore a high signal would be at say 5v or in some cases 24v. For Mach3 it is usually 5v when high, and 0v when low.
That being the case, the tick box next to the signals, asks you to define whether the signal is "active" when it is high or when it is low.
Therefore, if you were to change a signal in ports and pins to "active low" from "active high" it would cease to function as intended since it would be off when the software was expecting it to be on, or vice versa.
So to answer your question, yes you would detect that something was no longer working when using the machine
Regards
David