Figured it out - In Mach3 config > Port and Pins I had to tick Step Low Active... At that point the stepper was running at normal speed in one direction and really slowly in the other direction. Then realised I had incorrectly wired a ground needed by the routoutcnc motherboard (BoB). The motherboard has two GND pins and I was grounding one only (the wrong one). I switched it to the other GND and everything started working.

Thanks again for your help :)

This is a Routout cnc motherboard V2.1 and a Stepperonline DM542T driver and CNC4YOU 1.5Nm Nema23, on a Denford Novamill running Mach3 via a UC100 motion controller. I am using a stepper online 36V 400W power supply. The routout cnc motherboard is still powering the Y and Z axes - the new setup is currently for the X axis only.

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