To determine the prevalence of biased laboratory animal research designs in the published literature, we conducted a structured review of animal experiments published in North America and Europe in 2022 (Scientific Reports https://rdcu.be/eCisi). These studies involved sophisticated techniques, talented scientists, and thousands of laboratory animals. And yet, when we examined how the experiments were designed, we found a sobering truth: not even one followed the principles of rigorous experimental design.
At first, this was hard to believe. But the deeper we looked, the clearer it became: the flaws were not subtle details buried in statistical jargon. They were fundamental problems in how the experiments were set up in the first place.
Three Problems That Undermine Reliability
- Randomization and Blinding
In any valid experiment, chance must decide which subject goes into which group. Randomization helps ensure groups are balanced and comparable. And blinding protects against the human tendency — conscious or unconscious — to see what we expect to see. Yet in no study we reviewed were animals fully randomized, with all researchers and analysts completely unaware of the groups they were working with. That leaves results open to bias before the data are even analyzed.
2. The Cage Effect
Animals living together in the same cage influence each other and share the same micro-environment. No two cages are ever exactly alike. If one cage of animals is assigned to each treatment, then the effects of treatment and cage become inseparable. It’s like comparing two schools by testing just one classroom in each — you can’t tell whether differences come from the schools or the classrooms. This “completely confounded design” makes treatment effects impossible to assess.
A better solution is the Randomized Complete Block Design (RCBD), where animals from different treatment groups are mixed within cages. In this setup, each cage acts as a fair test, allowing valid comparisons between treatments while using fewer animals overall. If this is not possible, then a valid but less efficient and more expensive RCBD with multiple cages of animals assigned to each treatment can be used.
3. The Wrong Unit of Analysis
Finally, we found that many studies analyzed data as though each animal were independent, even when treatments were applied to entire cages. This practice, called pseudoreplication, is like baking one batch of cookies but pretending each cookie had a separate recipe. It artificially inflates the sample size and makes results look more convincing than they really are.
Why This Matters
These flaws undermine the credibility of the entire field of laboratory animal research. And the consequences ripple outward:
- Ethical costs: Millions of animals are used each year in research, with the expectation that their use will generate knowledge to advance health. If the designs are invalid, animals suffer without producing trustworthy science.
- Scientific and economic costs: Time, money, and talent are poured into experiments that cannot deliver reliable results.
- Societal costs: Treatments that look promising in flawed animal studies often fail in human trials — delaying progress and sometimes exposing volunteers to unnecessary risk.
It is no wonder that laboratory animal science faces a reproducibility crisis. Many of its roots can be traced to how experiments are designed.
The Way Forward
The solutions are not new. A century ago, statistician R. A. Fisher laid out the principles of sound experimental design: randomization, replication, and blocking. Applied correctly, these principles solve the problems we found. The Randomized Complete Block Design, in particular, explicitly accounts for cage effects, ensures random allocation, permits valid statistical analysis, and requires the fewest number of animals.
But technical fixes alone are not enough. Change must happen at every level:
- Researchers must design experiments using rigorous block designs, proper randomization, and blinding.
- Funding agencies and ethics boards must demand clear documentation of study designs and train reviewers to spot invalid ones.
- Journals must enforce standards that prevent fatally flawed experiments from being published.
- Universities must create graduate programs to train a new generation of experts in experimental design and analysis who can work alongside research teams, funding bodies, ethics committees, and editorial boards.
A Call to Action
Science depends on public trust. That trust is eroded when people learn that most animal experiments are invalid by design. By addressing these flaws, we can make animal research more ethical, efficient, and impactful. We can ensure that when animals are used, their contribution genuinely advances both human and animal health.
The tools to fix this are already in our hands. What’s needed now is the collective will to use them.
What do You Think?
Billions of dollars and millions of lives, human and animal, depend on the credibility of preclinical research. The tools to fix this problem already exist — what’s missing is the collective will to use them.
Our review shows that virtually all published comparative, laboratory animal studies suffer from biased designs. This conclusion is hard to dispute. But if anyone believes the current situation can be defended, we want to hear that defence — especially from funding agencies, research organizations, ethics boards, and journal editors, who are in the best position to mandate change.
And if no credible defence exists, then the harder questions remain: what must be done, and who will take responsibility?
We believe this calls for an open, frank discussion. What do you think? How can we, as a community, address these flaws and ensure that animal experiments truly deliver reliable knowledge?