Hello David,
This could be because you are trying to measure a very faint spectrum and ISIS is failing to detect the spectral trace in each image. If that is the case you should tick the box labelled Fixed Y value for sequence in the General tab and untick the box labelled Vertical coordinate Auto in the Calibration tab. Then manually adjust the contrast sliders at the bottom to make the spectrum visible in the Calibration tab and manually position the dotted red line in the Graticule over the spectrum. Then press Go and it should correctly locate the spectrum in each image and measure it correctly.
This assumes your guiding is good enough to keep the star at the same position in the slit (which corresponds to the Y coordinate in the calibration window). If the star is too faint to guide on successfully then the spectrum may be at a different Y coordinate in each image and the above recipe will not work. In that case you may have to put the target in the slit manually then guide on a brighter star in the guider window.
Hope that helps.
David