Biological time and in vivo research: a field guide to pitfalls

Biological rhythmicity is a fundamental characteristic of all life forms, from primitive bacteria to man. The molecular biology, genetics, and the neurobiology of the biological clock(s) are being elucidated. Daily (circadian) statistically significant fluctuations occur in all of the normal biological variables studied in the experimental animal and the human. Many researchers, however, are not aware of the negative impact biological rhythmicity can have on experimental design and/or data interpretation. This article serves not as a review, but as a "field guide" to the pitfalls that can occur when research is performed in the absence of an understanding of biological rhythmicity. The major topics discussed are: 1) data transfer from the diurnally in-active/resting/sleeping lab animal to the diurnally active human, 2) frequency of sampling, 3) free-running vs. synchronization, 4) alternating periods of resistance and susceptibility, 5) phase shifting of a rhythm, 6) the assumption that one mean +/- S.E. from control animals can be "stretched" across an experimental time span, and 7) plotting data on an "hours after treatment" format vs. a "time of day" format. The hope is that by avoiding the pitfalls, biological time will become an ally in the endeavor to understand human biology.