Learn how cage-level humidity and temperature shape thermogenesis, energy expenditure and metabolic outcomes in rodent studies. Real data, mechanisms and insights.

16.12.2025 – 6min

Micro-Environmental Humidity and Temperature in Rodent Metabolic Research

by Pierre-Louis Ruffault, PhD

Cage-level humidity and temperature in rodent metabolic research play a critical yet often underestimated role in shaping thermogenesis, energy expenditure, and metabolic outcomes. While ambient temperature is widely recognized as a key driver of metabolic rate in mice, increasing evidence shows that humidity and cage micro-environmental variability can substantially influence energy balance, obesity phenotypes, and experimental reproducibility.

Environmental parameters measured at the room level often fail to reflect what animals actually experience inside the cage. As a result, unrecognized micro-environmental differences may introduce variability that confounds metabolic and physiological readouts in preclinical studies.

Temperature, Thermoneutrality, and Chronic Cold Stress in Mice

Ambient temperature is a well-established determinant of rodent metabolism and obesity [1]. In mice, temperatures below thermoneutrality activate sustained thermogenic responses to maintain core body temperature, leading to increased whole-body energy expenditure [2], [3].

Standard laboratory housing temperatures (approximately 20–24 °C) therefore impose chronic cold stress. This condition elevates daily energy expenditure and alters not only metabolic but also immune outcomes compared with thermoneutral housing [4], [5]. Quantitative studies examining how small temperature shifts affect the ratio of daily energy expenditure to basal metabolic rate highlight the importance of carefully selecting and reporting housing temperatures in metabolic research [6].

Cold exposure further enhances sympathetic activation of brown adipose tissue (BAT) and thermogenic pathways, resulting in substantially higher resting and total energy turnover relative to thermoneutral conditions [3], [7].

Why Ambient Humidity Matters for Energy Balance

Beyond temperature, ambient humidity is emerging as another critical environmental cue influencing metabolic regulation. Mice possess sensory mechanisms capable of detecting changes in relative humidity (RH), triggering rapid physiological responses as well as longer-term adaptations that affect susceptibility to metabolic alterations.

Despite this, humidity often remains insufficiently regulated in laboratory vivariums. Variability in RH between facilities, or even within the same room, may therefore contribute to inconsistent metabolic outcomes across studies. Importantly, controlling temperature and humidity at the room level does not guarantee stable conditions at the cage level, where animals reside.

Cage-Level Micro-Environments and Hidden Variability

Significant heterogeneity in cage micro-environment temperature and humidity exists even under standardized housing conditions. Variations in RH and temperature within mouse cages frequently correlate with cage position within the rack, driven by differences in airflow, vertical temperature gradients, cage design, bedding materials, nesting behavior, and animal phenotype.

As a result, cages housed within the same room and rack may expose animals to markedly different micro-environmental conditions. These findings underscore a critical point: cage micro-environments are not equivalent, even when facility-level parameters appear tightly controlled.

In many laboratory settings, ventilation introduces air from the ceiling, creating a top-down airflow. Cages in the upper tiers are closer to air supply vents, resulting in increased air flow and lower RH due to enhanced moisture removal. Lower-tier cages often receive less airflow, trapping more humidity generated by respiration and bedding. 

Thermal gradients also play a role: warm air rises, making upper-tier cages warmer. Warmer air holds more moisture, which can decrease RH even when absolute humidity is constant. 

Empirical studies support these observations. Higher room ventilation rates (5 → 20 air changes per hour) reduce intra-cage humidity from 55% to ~36% RH, with the strongest effects in top-row cages [8], [9]. Even IVC systems – often viewed as a gold standard – show meaningful RH fluctuations despite >75 cage air changes per hour [10]. Importantly, these variations persist even when factors such as animal strain, cage temperature and feeding patterns are controlled. 

Moreover, even when apparent sources of variability such as cage temperature, animal genetic background, sex, and feeding behavior are controlled or appear homogeneous, notable differences in cage-level humidity still emerge. These observations underscore the importance of systematically considering microenvironmental humidity as a potential confounding factor when designing experiments and interpreting physiological or behavioral outcomes in rodents. 

Figure 1 below shows an example: RH measured inside cages varies systematically by cage level despite identical room conditions. 

Variations in temperature and RH can affect both animal welfare and experimental outcomes. Low RH induces a short-term increase in caloric intake (first 1–2 days), likely reflecting greater evaporative heat and water loss. High RH alters insulation and adipose tissue deposition [11]. Elevated humidity in lower-tier cages can promote ammonia accumulation [12], [13], causing respiratory irritation and welfare concerns, which then influence metabolic and behavioral data. 
 
Therefore, inconsistent environmental conditions across cages can introduce significant variability.  

PhenoMaster metabolic cage system for mice and rats with integrated sensors measuring temperature and humidity directly inside the rodent cage, enabling precise monitoring of micro-environmental conditions in metabolic research.

Why Measuring Temperature and Humidity at Cage Level Is Essential.

Room-level environmental monitoring does not capture the conditions animals actually experience. Measuring cage-level humidity and temperature allows researchers to:

• Detect micro-environmental gradients that alter thermogenic demand
• Reveal hidden sources of variability in metabolic phenotypes
• Identify cages with elevated ammonia accumulation and welfare risks
• Align environmental conditions across experimental replicates
• Improve reproducibility and interpretability of metabolic data
• Directly link environmental drift to changes in energy expenditure, food intake, and behavior

Even modest shifts in cage micro-environment – as little as 0.5–1 °C or a few percentage points of RH – can significantly influence metabolic readouts over time.

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Advanced Cage-Level Humidity Measurement

In-cage climate sensor integrated into the lid of a PhenoMaster metabolic cage, measuring humidity, temperature, and pressure inside the cage during mouse metabolic experiments.

In-Cage Climate Sensor

Animal welfare and data quality are intrinsically linked. PhenoMaster metabolic cages are designed to provide increased living space and uniform illumination while integrating climate sensors that measure humidity and temperature directly inside each cage.

This level of environmental precision minimizes experimental variability and ensures that critical micro-environmental cues – including humidity – are no longer overlooked in metabolic research.

Implications of Cage Micro-Environments for Obesity and Weight Gain


Experimental studies demonstrate that ambient humidity can directly influence weight gain and adiposity under high-fat diet conditions. For example, BALB/c mice housed at approximately 80% RH gained more weight and accumulated more fat than mice housed at approximately 20% RH. In these animals, brown adipose tissue retained more lipids, consistent with suppressed thermogenic activity [11].

Such findings support the concept that elevated humidity reduces thermogenic demand, conserves energy, and promotes fat storage. Importantly, these effects may be amplified when combined with high-calorie diets.

Effects on Metabolic Rate and Thermogenesis

Low relative humidity increases evaporative heat loss, forcing rodents to expend additional energy to maintain body temperature [11]. Conversely, high RH lowers thermogenic demand, conserves energy, and favors lipid storage [16].
Heat stress, often occurring in conjunction with high humidity, suppresses brown adipose tissue activity and mitochondrial function, further reducing energy expenditure [17]. Together, these mechanisms highlight how humidity interacts with temperature to shape metabolic rate and thermogenic capacity.

Food and Water Intake in Dry versus Humid Conditions

Rodents housed in low-humidity environments typically increase water intake and may transiently increase food consumption due to enhanced evaporative water loss [18]. However, this increased intake is often offset by higher energy expenditure.

In contrast, high humidity reduces water loss and energy expenditure. Under high-fat diet conditions, this energy conservation contributes to greater fat accumulation. Overall, differences in energy expenditure – rather than intake alone – appear to drive humidity-associated changes in body weight.

Beyond enabling thermoneutral housing or defined temperature challenges, the Climate Chamber also allows precise fine-tuning and stabilization of humidity. By maintaining stable humidity conditions, experimental variability is minimized, supporting reproducible metabolic research and enabling the investigation of subtle environmental cues that are often overlooked. 

Insulin Resistance and Glycemic Control

Short-term studies have shown no direct impairment in insulin sensitivity due to humidity alone. Experimental studies under high-temperature and high-humidity (HTH) conditions have shown increased adiposity and altered levels of metabolic hormones such as GLP-1 and ghrelin in mice, indicating early endocrine adaptations that may precede insulin resistance [19]. Although short-term studies report no clear change in insulin sensitivity due to humidity alone [20], the consistent association between high humidity, greater fat gain, and suppressed thermogenesis supports a model in which chronic exposure to humid environments could increase susceptibility to obesity and insulin resistance over time [11]. 

Rodent Strain Differences and Translational Relevance

Thermogenic and insulation responses vary substantially across rodent strains. Hairless mice exhibit stronger BAT activation under cold exposure [21], while animals with thinner dermal adipose layers require greater thermogenic output [22].

Because mice are small and lose heat rapidly, precise control of temperature and humidity is particularly critical [23]. These principles have parallels in humans, where environmental humidity and temperature influence thermal comfort, energy expenditure, and metabolic regulation [24]. 

Conclusion

Cage-level humidity and temperature are critical biological variables that shape thermogenesis, energy expenditure, and metabolic phenotypes in rodent studies. Failure to measure and control these parameters at the cage level risks introducing hidden variability that undermines reproducibility and translational relevance.
Integrating cage-level environmental monitoring and control represents a necessary step toward more accurate and reproducible metabolic research.

References and Key Studies 

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C. J. Gordon, “Thermal physiology of laboratory mice: Defining thermoneutrality,” J. Therm. Biol., vol. 37, no. 8, pp. 654–685, Dec. 2012, doi: 10.1016/j.jtherbio.2012.08.004.

B. Cannon and J. Nedergaard, “Brown adipose tissue: function and physiological significance,” Physiol. Rev., vol. 84, no. 1, pp. 277–359, Jan. 2004, doi: 10.1152/physrev.00015.2003.

[4] A. W. Fischer, B. Cannon, and J. Nedergaard, “Optimal housing temperatures for mice to mimic the thermal environment of humans: An experimental study,” Mol. Metab., vol. 7, pp. 161–170, Oct. 2017, doi: 10.1016/j.molmet.2017.10.009.

K. M. Kokolus et al., “Baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature,” Proc. Natl. Acad. Sci. U. S. A., vol. 110, no. 50, pp. 20176–20181, Dec. 2013, doi: 10.1073/pnas.1304291110.

J. Keijer, M. Li, and J. R. Speakman, “What is the best housing temperature to translate mouse experiments to humans?,” Mol. Metab., vol. 25, pp. 168–176, Apr. 2019, doi: 10.1016/j.molmet.2019.04.001. 

H. Sell, Y. Deshaies, and D. Richard, “The brown adipocyte: update on its metabolic role,” Int. J. Biochem. Cell Biol., vol. 36, no. 11, pp. 2098–2104, Nov. 2004, doi: 10.1016/j.biocel.2004.04.003.

C. K. Reeb, R. B. Jones, D. W. Bearg, H. Bedigian, and B. Paigen, “Impact of Room Ventilation Rates on Mouse Cage Ventilation and Microenvironment,” Contemp. Top. Lab. Anim. Sci., vol. 36, no. 1, pp. 74–79, Jan. 1997.

G. M. Ward, K. Cole, J. Faerber, and F. C. Hankenson, “Humidity and Cage and Bedding Temperatures in Unoccupied Static Mouse Caging after Steam Sterilization,” J. Am. Assoc. Lab. Anim. Sci. JAALAS, vol. 48, no. 6, pp. 774–779, Nov. 2009, Accessed: Nov. 12, 2025. [Online]. Available: https://pmc.ncbi.nlm.nih.gov/articles/PMC2786932/

Techniplast, “Monitoring of environmental conditions in Tecniplast Emerald EM500 Individually Ventilated Cages.”

I. Kasza et al., “‘Humanizing’ mouse environments: humidity, diurnal cycles and thermoneutrality,” Biochimie, vol. 210, pp. 82–98, July 2023, doi: 10.1016/j.biochi.2022.10.015. 

M. D. Rosenbaum, S. VandeWoude, and T. E. Johnson, “Effects of Cage-Change Frequency and Bedding Volume on Mice and Their Microenvironment,” J. Am. Assoc. Lab. Anim. Sci. JAALAS, vol. 48, no. 6, pp. 763–773, Nov. 2009, Accessed: Nov. 12, 2025. [Online]. Available: https://pmc.ncbi.nlm.nih.gov/articles/PMC2786931/

M. D. Rosenbaum, S. VandeWoude, J. Volckens, and T. E. Johnson, “Disparities in Ammonia, Temperature, Humidity, and Airborne Particulate Matter between the Micro-and Macroenvironments of Mice in Individually Ventilated Caging,” J. Am. Assoc. Lab. Anim. Sci. JAALAS, vol. 49, no. 2, pp. 177–183, Mar. 2010, Accessed: Nov. 12, 2025. [Online]. Available: https://pmc.ncbi.nlm.nih.gov/articles/PMC2846005/

H. A. Paz et al., “Impact of short-term housing temperature alteration on metabolic parameters and adipose tissue in female mice,” Front. Endocrinol., vol. 16, June 2025, doi: 10.3389/fendo.2025.1617262.

Y. Wu et al., “Gut microbiota associated with appetite suppression in high-temperature and high-humidity environments,” EBioMedicine, vol. 99, p. 104918, Jan. 2024, doi: 10.1016/j.ebiom.2023.104918.

I. Kasza et al., “Evaporative cooling provides a major metabolic energy sink,” Mol. Metab., vol. 27, pp. 47–61, Sept. 2019, doi: 10.1016/j.molmet.2019.06.023.

P. Lai et al., “Heat stress reduces brown adipose tissue activity by exacerbating mitochondrial damage in type 2 diabetic mice,” J. Therm. Biol., vol. 119, p. 103799, Jan. 2024, doi: 10.1016/j.jtherbio.2024.103799.

P. Ke, “The effect of relative humidity on water intake of C57BL/6J mice housed under conditions of controlled relative humidity at cage level”.

S. Chen et al., “High temperature and humidity in the environment disrupt bile acid metabolism, the gut microbiome, and GLP-1 secretion in mice,” Commun. Biol., vol. 7, no. 1, p. 465, Apr. 2024, doi: 10.1038/s42003-024-06158-w.

H. Pallubinsky et al., “Passive exposure to heat improves glucose metabolism in overweight humans,” Acta Physiol. Oxf. Engl., vol. 229, no. 4, p. e13488, Aug. 2020, doi: 10.1111/apha.13488.

J. Houstĕk and M. Holub, “Cold-induced changes in brown adipose tissue thermogenic capacity of immunocompetent and immunodeficient hairless mice,” J. Comp. Physiol. [B], vol. 164, no. 6, pp. 459–463, 1994, doi: 10.1007/BF00714583.

I. Kasza, D. Hernando, A. Roldán-Alzate, C. M. Alexander, and S. B. Reeder, “Thermogenic profiling using magnetic resonance imaging of dermal and other adipose tissues,” JCI Insight, vol. 1, no. 13, Sept. 2016, doi: 10.1172/jci.insight.87146.

C. M. James, S. H. Olejniczak, and E. A. Repasky, “How murine models of human disease and immunity are influenced by housing temperature and mild thermal stress,” Temp. Multidiscip. Biomed. J., vol. 10, no. 2, pp. 166–178, doi: 10.1080/23328940.2022.2093561.

C. F. Juna, Y. H. Cho, D. Ham, and H. Joung, “Associations of Relative Humidity and Lifestyles with Metabolic Syndrome among the Ecuadorian Adult Population: Ecuador National Health and Nutrition Survey (ENSANUT-ECU) 2012,” Int. J. Environ. Res. Public. Health, vol. 17, no. 23, p. 9023, Dec. 2020, doi: 10.3390/ijerph17239023.


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