Table 3.

Studies that utilised grimace scoring in the dark stage of the circadian cycle and the impact on scores were reported.

Reference.	Reporting Detail	Intervention	Compared with Measures in Light (Y/N)	Direction of Effect for Comparison between Light and Dark
[51]	Conducted circadian study comparing light and dark recordings	Ventral ovariectomy and response to analgesics	Y	Compared mice which had surgery in the morning versus the evening with measurement timepoints of baseline, and every 6 h past surgery for 48 h There was no circadian effect on baseline MGS scores. However, mice operated on in the morning displayed larger MGS increases 12 h after surgery compared to 24 h, whilst mice operated on in the evening showed smaller increases at these time points. This suggests that mice experience higher levels of postoperative pain at night (dark phase)
[54]	No dark cycle recording but did compare MGS across the light phase	Biological	N	Live scoring There was no difference in MGS score between three time points (9 am, 12.30 pm, 4 pm) for C57BL/6, CD-1 or C3H/He mice. BALB/c mice showed a greater score at Noon compared to AM. Retrospective Scoring There was no significant difference in MGS scores between the three time points for CD-1, C3H/He or BALB/c mice. C57BL/6 mice showed a greater MGS scores at both Noon and PM time points compared to the AM time point
[60]	Performed a restrained grimace technique in dark (manipulated dark condition- not part of cycle)	CGRP-induced migraine	Y (compared bright light)	Grimace scores were higher in the dark than in bright light for the CD1 mice. Light transition led to decreased orbital tightening and nose bulge. C57BL/6J mice showed no significant difference between the CGRP-induced grimace in light and dark. Responses to CGRP were generally similar in direction as those recorded in the light.