Mean Warming Misses the Point: Including Diel Thermal Variability as an Essential Practice in Aquatic Warming Experiments

Understanding how climate warming alters aquatic ecosystems requires accurately representing the thermal environments that organisms experience. Yet a fundamental methodological limitation persists across most warming experiments. Most manipulative studies simulate future warming using constant temperature offsets relative to ambient conditions, implicitly assuming that increases in mean temperature adequately represent the ecological consequences of climate change on aquatic ecosystems. However, significant diel thermal variation is intrinsic to virtually all inland aquatic and coastal ecosystems, emerging from the daily cycle of solar heating, radiative cooling, meteorological forcing, hydrology, and mixing dynamics. Aquatic organisms thus inhabit thermal environments characterized by continuous fluctuation around a shifting baseline, not static temperature elevation. We argue that aquatic warming experiments must systematically incorporate realistic diel thermal variability to adequately represent the ecological consequences of climate change, particularly as heatwaves are projected to become more frequent and intense in aquatic ecosystems. Aquatic experiments that ignore this variability inevitably constrain the range of physiological, metabolic, and trophic responses available for detection. Although diel variation occurs across latitudes and inland aquatic ecosystem types, its ecological consequences are not uniform. In many systems diel variation is modest and buffered by depth, mixing, groundwater inputs, or seasonal stratification. In others, especially small shallow water bodies, limited thermal inertia produces large amplitude oscillations (Woolway et al. 2016). Recent observations from Amazonian lakes demonstrate these extreme end of this continuum: temperatures exceeding 40°C and diel oscillations reaching 13°C were recorded during a heatwave event (Santos Fleischmann et al. 2025). These conditions reveal how rapidly tropical waters can heat and cool, and how limited thermal buffering exposes organisms to wide temperature ranges within short intervals. Similar processes operate in temperate systems during warm summers, drought years, and in hydrologically isolated ponds, wetlands, reservoirs, and low-order streams. Even large lakes experience diel thermal dynamics in surface mixed layers and are also undergoing severe heatwaves (Yang et al. 2025). However, in these systems thermal amplitudes tend to be smaller due to greater water volume and deeper circulation (Woolway et al. 2016). Therefore, as reported by Santos Fleischmann et al. (2025), the interaction between high mean temperature, limited depth, high solar exposure, turbidity, and restricted mixing makes small tropical lakes the systems most likely to experience ecologically consequential thermal instability. Such systems lack deep cold refuges, maintain narrow oxygen budgets, and sustain communities dominated by ectotherms operating close to upper thermal tolerance limits (Nati et al. 2021). Ignoring diel thermal variation of inland and coastal aquatic systems has profound implications for interpreting their ecological responses to warming. Temperature-dependent biological rates are often non-linear. According to Jensen's inequality, nonlinear functions cannot be represented by their mean (Fig. 1). Metabolic and physiological responses therefore depend not only on average conditions but also on the amplitude and frequency of temperature fluctuations. A system fluctuating between 15°C and 35°C cannot be approximated by a system held at constant 25°C even though the arithmetic mean is identical. High temperature excursions disproportionately elevate respiration and oxygen demand while oxygen availability declines. Repeated exposure to such extremes generates cumulative metabolic stress invisible in constant offset experiments (Rezende and Carter 2024). These nonlinearities can have effects propagating from individuals to ecosystems. Larger consumers are especially vulnerable because body mass scales with higher absolute oxygen requirements and narrower thermal safety margins (Rubalcaba et al. 2020). Repeated passage through high temperature phases increases susceptibility to hypoxia and reduces aerobic scope, constraining foraging, growth, and movement. Smaller organisms, with higher surface area to volume ratios and lower oxygen demand, are less susceptible to the same fluctuations because their lower absolute metabolic costs and more efficient oxygen diffusion buffer them against transient thermal stress. Diel thermal instability therefore provides a mechanistic basis for the well-known global pattern that warming tends to favor smaller taxa. This mechanism may be especially strong in tropical freshwaters where large amplitude thermal cycles and high baseline temperatures combine to compress food web structure, reduce representation of high trophic levels, and strengthen the dominance of microbial and detrital pathways (O'Gorman et al. 2012; Dantas et al. 2019). Ecosystem metabolism is also sensitive to thermal variability (Yvon-Durocher et al. 2010). Respiration increases more steeply with temperature than gross primary production. Short periods at very high temperatures thus amplify respiratory losses more strongly than they enhance photosynthesis. Nocturnal cooling does not fully compensate for daytime metabolic surges, particularly when nighttime oxygen levels remain low. Many tropical lakes are shallow, turbid, and exposed to intense solar radiation, conditions that enhance heat absorption, limit mixing depth, and maintain chronically low oxygen availability. When diel thermal variation interacts with drought-driven shallowing or stratification instability, the likelihood of net heterotrophy, carbon mineralization, and elevated CO2 emissions increases. Climate change is expected to intensify heatwaves and reduce water levels in many regions, conditions that further increase diel thermal amplitude. Extreme thermal events are becoming more common across lake size classes and latitudes (Woolway et al. 2025). The ecological consequences of ignoring diel variability in experimental designs therefore extend well beyond the tropics. This scenario underscores a methodological gap in climate warming research. Constant offset experiments constrain the thermal experience of organisms to narrow ranges and uniform rates of change. This design simplifies replication and control but fails to represent the physical reality of natural environments. Experimental outcomes may thus systematically underestimate the magnitude and diversity of warming impacts and misrepresent mechanisms by obscuring nonlinear responses, threshold effects, and interactions between temperature, oxygen, and metabolic demand. Addressing this gap requires incorporating temperature variability into experimental and modeling frameworks. Fluctuating temperature treatments that mimic natural diel cycles can be implemented with programmable heaters, infrared systems, or variable thermal control algorithms. High-frequency thermal monitoring should accompany these experiments to verify that thermal dynamics approximate natural conditions. Space-for-time substitutions offer a complementary approach: by comparing ecosystems distributed along natural temperature gradients, it is possible to capture realistic diel thermal variation and long-term acclimatization, though at the cost of reduced experimental control. Combining these approaches with mechanistic models that integrate full temperature distributions rather than average values can help clarify how thermal variability modifies metabolic, physiological, and ecosystem-level responses to warming. Diel thermal instability should be recognized as a primary dimension of climatic change in aquatic ecosystems. It is a pervasive physical driver with profound ecological implications. Tropical shallow lakes represent the systems where this instability is most extreme, but the underlying mechanisms operate globally. Because warming tends to increase diel thermal variability, aquatic warming experiments that span different seasons should incorporate the seasonal changes in the magnitude of daily fluctuations, with larger fluctuations expected during warmer periods. To understand how warming reshapes aquatic biodiversity, trophic structure, and carbon cycling, experimental and observational studies must confront the full complexity of thermal environments. Mean temperature alone is no longer an adequate descriptor of warming in aquatic systems. The Article Processing Charge for the publication of this research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (ROR identifier: 00x0ma614).