What time of day do you perform behavioral assays in mice?


Is there a right or better time to test your animals in nociceptive behavioral assays?

Some people say morning, some say afternoon. What do you think?

From my experience, the mice seem to be the most subdued (chill, quiet, not fidgety) in the afternoon. But that may just be because they have had more time to acclimate to the testing environment. For example, for von Frey, I put the mice in their containers around 9 am, and then by 12 pm they seem to get quiet. But it takes a few hours.

A recent paper by Minett et al. actually looked at this, von Frey thresholds at different times of the day, and found significant diurnal variation. In the early afternoon (1-3 PM), the mice have the highest mechanical thresholds.

Minett figure
(From Minett et al. 2014)

If this is true, it would have implications for all of our behavior experiments and in particular, time course experiments.

Perhaps the best we can achieve is consistency. Test the same time every day. But when you do a battery of assays, or if assays stretch across the whole day (a time course of von Frey), then there is no avoiding the potential drift in behavioral responses due to circadian rhythm or whatever.

@LegakisL @ram_kandasamy @janehartung @tberta @jmogil @fmoehring


All of my von Frey testing is done in the hour before the start of the dark phase (4-5 pm). I’ve also done von Frey testing at 9-10 am. There was no noticeable difference in mechanical or thermal withdrawal thresholds in those groups. I think that’s primarily because CFA causes robust inflammation that any change is likely washed out.

My lab has previously explored hot plate latencies over the circadian rhythm, which remained unchanged when rats were tested every 6 hours. I like your suggestion - consistency is likely the key. Time courses certainly are challenging; however, things should work out as long as there is an appropriate control group. I’m not sure if anyone has examined potency changes of analgesics over the circadian rhythm, but that can certainly could pose a problem.


All of my von Frey and other behavior assays are performed between 8-1pm. And I try to be very consistent between the time of the day I perform each behavior assay.
I think this once again should just be something thats reported in the methods section of a paper, so that if you do see differences between your own behavior and someone else’s, its easier to figure out why you might see those differences.


Thanks for the info @fmoehring Do you assay during that time for any particular reason, whether it be convenience or something you noticed about the mouse’s behavior?

On a related note, because of your paper, I’m paying attention now to the bedding material. Thanks for such careful observation and most importantly, for taking the time to report it! This is the kind of thing that people don’t pay attention to, but that can make a big difference.


Thanks for chiming in @ram_kandasamy

Things like CFA or SNI are so dramatic, that you’re already near the lower limits of detection anyway, and the variance really becomes small. I think there is a lot more variability in naive animals, in fact. In any case, consistency, as best as one can.


Yes we have noticed that our mice settle a lot faster in the morning times, and are therefore sitting a lot calmer than when we performed the experiments in the afternoon. Our mice were jumpier when we performed the experiments later in the day.

And thank you very much for your kind words about my paper. Its just one sentence in the methods section but could make a difference when someone is trying to replicate your data and can’t do it in the end.


Morning, always. If only because as everyone has mentioned the mice seem to settle faster and this allows me to have a consistent acclimation time.


@lfqueme Yeah, everyone says they’re quiet in the morning. Idk. When I first bring them into the behavior room, they’re jumping around for at least two hours. We usually do behavior right after lunch, around 1-3 pm. That seems to be a sweet spot. Also, note the diurnal variation from Minett et al. above. Midday seems to be when they have the highest paw withdrawal threshold.


I would say as long as you keep it constant within your experiments the time might be something worth noting in the methods but I would go with what works best for you and your laboratory.