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I would like to analyze repeatedly measured outcome (20 measures) data from an experimental trial I am running on three mice ($n=3$). My dependent variable is continuous but not normally distributed (and it is resistant to all forms of transformation), so I was looking for alternatives to the repeated measures of ANOVA. From my understanding, the GEE method does not require the dependent variable to be normally distributed, but needs a larger number of observations.

Question: Could you give me advice for a statistical analysis method I could use?

enter image description here

According to the comments and suggestions to my initial question, I have followed following steps:

I have set my previous dependent variable as independent variable (continuous predictor) and created a binary variable which would be my dependent variable and would represent the occurrence of an event (yes/no).

My hypothesis is that higher values of my independent variable are associated with the occurrence of a certain event.

event_c= binary outcome, p1= independent continuous predictor, 
Fixed effect = p1, Random effect= mouse

So I have set following command in R:

glmer(event_c~p1+mouse+(p1|mouse), data=mydata1, family="binomial")

And here is the output:

enter image description here

Although the p-values show a statistically significant results, I am a little bit confused about the values of the intercept and was wondering if my results are plausible and whether my approach is correct.

(I have tried to solve a simple logistic regression and again the intercept values would then give me a very low odds ratio)

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  • $\begingroup$ Welcome to Cross Validated. Can you give us an idea of the distribution of your continuous dependent variable? Screenshots of the histogram would be helpful. $\endgroup$ Commented May 3, 2016 at 23:09
  • $\begingroup$ Thanks for editing my post. I have added the histogram of my distribution. $\endgroup$
    – a.pardew
    Commented May 4, 2016 at 0:12
  • $\begingroup$ What is the dependent variable? Do you need to keep it as continuous or can you dichotomize/categorize it somehow? $\endgroup$ Commented May 4, 2016 at 0:15
  • $\begingroup$ The dependent variable is the activating function (it is a measure of the influence of an extracellular field on axons/neurons). I could perhaps categorize the values in either negative/positive (as negative values are associated with hyperpolarization whereas positive values are associated with depolarization). Would that help? $\endgroup$
    – a.pardew
    Commented May 4, 2016 at 0:36
  • $\begingroup$ That would help, as long as the results would be meaningful to your field. If it doesn't make sense to do it, then don't. Check the literature to see what others have done with the dependent variable $\endgroup$ Commented May 4, 2016 at 1:01

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Most of the issues in the original question and its trail of comments arise from only having 3 animals in the data set. When I was doing animal experiments, long ago, there was a rule-of-thumb that you should have at least 6 animals per group. It thus may be hard in any event to get results published based on 3 cases. Nevertheless, let's see what can be done with this limited data set.

As I understand the setup, there is an experimentally applied "escalating dose of a specific parameter," which alters the "activating function" ("a measure of the influence of an extracellular field on axons/neurons"), which in turn is thought to influence the probability of occurrence of some event. The displayed histogram is for the "activating function" values. There is a total of 39 observations of (event/no-event) among 3 animals, according to the output from the logistic regression attempt, although there seem to be a few more observations of the activating function in the histogram.

The "activating function" in this case would be a "dependent variable" from the perspective of the applied parameter, but under the logic of the previous paragraph the "activating function" would be considered a predictor variable for the event. To analyze the data this way you would have to know (or demonstrate) that the applied parameter only influenced the event probability through its influence on the "activating function." We'll put that aside for now.

For predicting event occurrence, the rule of thumb is that you need about 15 of the least-frequent of (event/no-event) per predictor variable examined in a logistic regression model. With 39 observations, that number can be no more than 19. That only leaves room for 1 predictor variable without danger of over-fitting. So one possibility would be logistic regression of event against the "activating function" (called "p1" in the displayed regression output). The significance of the regression slope coefficient for p1 in the logistic regression could be evaluated, although with so few total observations the estimate of the intercept will tend to be imprecise.

With more events among the 3 mice, or a larger number of mice, it might be possible to include a correction for mouse-to-mouse differences. The original idea in the question was to treat mice as a random effect (1|mouse). This has the advantage of only counting as 1 extra predictor in the model, but doesn't always work well with so few mice. Treating the 3 mice as fixed effects would be equivalent to adding 2 predictors to the model, which is more than the present data set might be able to handle reliably. Including interactions or random effects for both the intercept and regression slopes would require even more cases.

Another way to proceed would be to compare the activating-function values associated with the events versus the no-events in a linear model. That is effectively treating the activating function values as a "dependent" variable, with the event/no-event as the "independent" variable, even though the logical/causal direction is opposite. There's nothing wrong with that. If significant differences were found, one could say "The occurrence of an event was associated with a higher activating-function value..." With 39 observations you could handle 2 or maybe even 3 predictors in such a linear model.

This brings us back pretty much to the original idea in the question: treat the "activating function" as a dependent variable, with mice perhaps considered as a random effect or fixed effects. This data sample probably would allow for evaluating that number of predictors, although the small number of animals would make one worry abut its applicability to other sets of the same mice, other strains of mice, other related but not identical experimental conditions, and so forth.

This analysis would not require a normal distribution of the observations. For properly interpreting p-values and the like what you need is normal distributions of the residual errors around the fitted values for the groups analyzed. Unfortunately, the way some introductory statistics course are taught can lead to a misplaced emphasis on normal distributions of the data themselves. Non-parametric tests could also be considered if residuals are non-normal.

Finally, back to the logic of the experimental setup. If you are manipulating some "parameter" that affects the "activating function" and then the probability of the event changes, you really need to make sure that the "activating function" value is not just an epiphenomenon that happens to change along with a direct effect of the "parameter" on the "event" probability. Yes, you can always make the association between the "activating function" and the event, by logistic regression or a linear model, but the causal interpretation requires a good deal of backup in this type of experimental design.

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  • $\begingroup$ Thanks @EdM for your very precise answer! It's very helpful! Though, just as a matter for clarification for me: when you talk about linear model, are you thinking about a repeated measures anova using the lme function in r (in my case: "model1<-lme(p1~event_c, random=~1|mouse/event_c, data=mydata1, method="ML") where p1 the activating function? I suppose I can't use a simple linear regression (lm) as my data observations are not independent? $\endgroup$
    – a.pardew
    Commented May 14, 2016 at 20:29
  • $\begingroup$ Yes, that's the idea, although I confess that I have a lot of trouble with the detailed coding of random effects in the function calls. You should account in some way for influences from individual mice, and including a random effects term for them could do that. Typically you should have more mice to get an estimate of the variance due to mice, but it might work well enough for your purpose. With nearly 40 cases you might get by OK with treating them as fixed effects instead; only 3 predictors then (event, 2 for the 3 mice). $\endgroup$
    – EdM
    Commented May 14, 2016 at 23:50
  • $\begingroup$ Brilliant! Thank you very much EdM for your help during this process. BR A. Pardew $\endgroup$
    – a.pardew
    Commented May 15, 2016 at 18:49

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