GraphPad Statistics Guide

Advice: Don't keep adding subjects until you hit 'significance'.

Advice: Don't keep adding subjects until you hit 'significance'.

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Advice: Don't keep adding subjects until you hit 'significance'.

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A commonly used approach leads to misleading results

This approach is tempting, but wrong (so shown crossed out):

Rather than choosing a sample size before beginning a study, simply repeat the statistical analyses as you collect more data, and then:

If the result is not statistically significant, collect some more data, and reanalyze.

If the result is statistically significant, stop the study.

The problem with this approach is that you'll keep going if you don't like the result, but stop if you do like the result. The consequence is that the chance of obtaining a "significant" result if the null hypothesis were true is a lot higher than 5%.

Simulations to demonstrate the problem

The graph below illustrates this point via simulation. We simulated data by drawing values from a Gaussian distribution (mean=40, SD=15, but these values are arbitrary). Both groups were simulated using exactly the same distribution. We picked N=5 in each group and computed an unpaired t test and recorded the P value. Then we added one subject to each group (so N=6) and recomputed the t test and P value. We repeated this until N=100 in each group. Then we repeated the entire simulation three times. These simulations were done comparing two groups with identical population means. So any "statistically significant" result we obtain must be a coincidence -- a Type I error.

The graph plots P value on the Y axis vs. sample size (per group) on the X axis. The green shaded area at the bottom of the graph shows P values less than 0.05, so deemed "statistically significant".

Experiment 1 (green) reached a P value less than 0.05 when N=7, but the P value is higher than 0.05 for all other sample sizes. Experiment 2 (red) reached a P value less than 0.05 when N=61 and also when N=88 or 89. Experiment 3 (blue) curve hit a P value less than 0.05 when N=92 to N=100.

If we followed the sequential approach, we would have declared the results in all three experiments to be "statistically significant". We would have stopped when N=7 in the first (green) experiment, so would never have seen the dotted parts of its curve. We would have stopped the second (red) experiment when N=6, and the third (blue) experiment when N=92. In all three cases, we would have declared the results to be "statistically significant".

Since these simulations were created for values where the true mean in both groups was identical, any declaration of "statistical significance" is a Type I error. If the null hypothesis is true (the two population means are identical) we expect to see this kind of Type I error in 5% of experiments (if we use the traditional definition of alpha=0.05 so P values less than 0.05 are declared to be significant). But with this sequential approach, all three of our experiments resulted in a Type I error. If you extended the experiment long enough (infinite N) all experiments would eventually reach statistical significance. Of course, in some cases you would eventually give up even without "statistical significance". But this sequential approach will produce "significant" results in far more than 5% of experiments, even if the null hypothesis were true, and so this approach is invalid.

Bottom line

It is important that you choose a sample size and stick with it. You'll fool yourself if you stop when you like the results, but keep going when you don't. The alternative is using specialized sequential or adaptive methods that take into account the fact that you analyze the data as you go. To learn more about these techniques, look up 'sequential' or 'adaptive' methods in advanced statistics books.