Figure 3. Four Protocols for Investigating Circadian Rhythms in Humans.

(A) The Constant Routine Protocol involves a greater than 24-hour period of constant wakefulness wherein all external factors (including light, posture, feeding, and temperature) are kept constant. This protocol allows reconstruction of the entire circadian rhythm but does not enable investigation of circadian misalignment. (B) The Inverted Sleep-Wake Cycle involves periods of nocturnal and daytime sleep, separated by a prolonged period of wakefulness. Feeding and posture are typically kept constant throughout the protocol, but light levels are set to match changes in sleep/wakefulness. This protocol provides insight into both circadian and behavioral cycles. (C) The Circadian Alignment/Misalignment Protocol includes two subprotocols: the alignment protocol with daytime behavioral cycles occurring as they would normally, and a misaligned protocol, with those identical behavioral cycles occurring during the biological night. Light levels are varied, and participants eat normal meals and snacks. While this protocol does not allow reconstruction of the underlying circadian rhythm, it does reveal how much a diurnal rhythm is influenced by the circadian phase (i.e., the time of day), circadian misalignment, and behavioral factors. (D) The Forced Desynchrony Protocol involves following 20- or 28-day hour days for typically 1–2 weeks to cycle through different alignments between circadian rhythms and behavioral rhythms. Light levels during wakefulness are kept very low, and participants consume normal meals and snacks. Mathematical procedures are then used to extract the underlying circadian versus behavioral components of the diurnal rhythm. This protocol also reveals the impact of circadian misalignment on biologic endpoints.