Refined Mouse Handling Publications

There have been an increasing number of peer-reviewed publications investigating refined mouse handling. At least 19 peer-reviewed publications support the use of refined mouse handling. They find that refined mouse handling improves mouse welfare and scientific quality.

Key paper findings include that refined mouse handling:

• Reduces anxiety as measured by elevated plus maze, social novelty test, open field test, and light-dark box test 3, 5, 6, 8, 11, 12, 13, 18, 21, & 22 
• Reduces depressive-like behavior as measured by sucrose reward, resilience to negative events, forced swim test and burrowing test 5, 7, 12
• Reduces chronic stress as measured by adrenal gland size 5 
• Increases test reliability 13 
• Improves physiological parameters such as improving glucose tolerance & reducing blood glucose & corticosterone 17, 18 
• Increases voluntary interaction with the handler 4, 5, 6, 7, 10, 13, 14, 21 
• Improving breeding as measured by larger pups, more pups born & weaned, and a longer breeding productive lifespan 1 

You can find a list of the publications cited here and a brief description of their findings below. For a more extensive summary of each paper’s methods and findings, see NC3Rs mouse handling research summary. 

Brief publication descriptions:

1. Luchins & Felgenhauer 2026. Enhancing Rodent Welfare, Cost Savings, and Efficiency: Implementation and Review of Data-Driven Improvements. Journal of the American Association for Laboratory Animal Science. DOI:  10.30802/AALAS-JAALAS-25-170

• Outlines institutional approach of enhancing animal welfare and operational practices in a rodent program, including replacing tail handling with refined methods (tunnel and cupping)
• Tunnel handling was implemented in ~30% of mouse cages and integrated with home‑cage enrichment, giving mice choice and control during handling while also supporting staff ergonomics, reducing bite risk, and improving morale and retention among animal care personnel.
• Practical refinements supported scalability and cost‑effectiveness, including repurposing unused water bottles into handling tunnels, enabling broader adoption of refined handling without added expense while maintaining welfare and operational benefits.
  

2. Otsuka et al. 2026. Effects of scruff restraint and handling–tail and tunnel–on plasma corticosterone in female BALB/c mice (Mus musculus). Journal of the American Association for Laboratory Animal Science. DOI: 10.30802/AALAS-JAALAS-25-134

• Scruff restraint (single and repeated) increased plasma corticosterone in both tail- and tunnel-handled mice, but the handling method did not significantly impact plasma corticosterone concentrations following restraint
• Tunnel-handled mice showed increased voluntary interactions with the handler compared to tail-handled mice, even when scruff-restrained
• These findings suggest that the handling method has minimal impact on physiologic stress following brief restraint, supporting the use of less-aversive handling techniques without concern for confounding stress effects
  

3. Schlutt et al. 2026. A novel application tunnel in combination with medical training reduces stress induced by frequent intraperitoneal injections and blood sampling in mice. BioRxiv. DOI: 10.64898/2026.01.08.698459

• In a mouse model of hepatocellular carcinoma, the use of a novel “application tunnel” for restraint during IP injections reduced stress during IP injections, as demonstrated through decreased latency to voluntary interaction, reduced Mouse Grimace Scale scores, reduced defecation/urination, and faster return to normal activity when compared to mice scruffed during IP injections
• Mice were acclimated to the application tunnel using methods similar to those used to acclimate mice to tunnel-handling
• The acclimation tunnel was also used in non-HCC mice undergoing blood draws, and similar benefits were seen
  

4. Bodnar et al. 2025. The effects of handling on mouse behavior: cupped hands versus familiar or novel huts or tunnels. PLoS ONE. DOI: 10.1371/journal.pone.0323785

• Handling via mouse igloos (huts) may be a refined handling method, with higher voluntary interaction times than tunnel-handling or cupped-handling, and similar rates of exploration of an elevated plus maze
• Although cage familiarity with the igloo (and tunnel) improved voluntary interaction at the outset, no differences were evident by day 5
  
  

5. Meyer et al. 2025. Lack of evidence for a consistent differential impact of tail and tunnel handling on markers of welfare in laboratory mice. Scientific Reports. DOI: 10.1038/s41598-025-07384-w

• Argues that most tunnel handling literature investigates the effect of handling performed in an unusually high frequency and prolonged duration, not accurately matching laboratory routines; BUT, ignores certain important aspects of the literature:
• Ignored findings of Gouveia & Hurst 2019: Handling for only 2 seconds during cage changes is sufficient to familiarize mice with tunnel handling (cage change every 2 weeks) and see benefits
• Did not provide the tunnel inside of the cage as enrichment, and only introduced for handling – thus, ignored Gouveia & Hurst 2013 findings that use of a familiar tunnel that stays in the cage sees greater benefits
• In voluntary interaction, mice in both tail- and tunnel-handled groups were offered interaction with the handler’s hand, NOT the tunnel, inconsistent with the refined handling literature
  
  

6. Hohlbaum et al. 2024. The implementation of tunnel handling in a mouse breeding facility revealed strain-specific behavioral responses. Laboratory Animals. DOI:  10.1177/00236772231215077 

• When implemented once weekly during cage change in a breeding unit with three inbred strains, tunnel-handled mice defecated less during handling than tail-handled mice
• One strain (NZW) saw further benefits: increased voluntary contact while handling, decreased hiding in an enrichment house before handling, and increased exploration of the cage pre-handling; results in two other strains (Hello Kitty and WNK) were more ambiguous
• Tunnel-handling took 3 seconds longer on average than tail-handling, but became faster over time in the NZW strain

7. Swan et al. 2024. Refinement by gentle handling of mice affects oral-dosing pharmacokinetic end points and response to stress under drug administration and sampling. Pharmacological Research. DOI: 10.1016/j.prerep.2024.100020

• Mice habituated to tunnel-handling had higher maximum serum concentration (C_max), lower time to C_max(T_max), and 30% higher drug exposure than mice who were tail-handled or tunnel-handled without habituation, indicating decreased stress and improved drug uptake
• These findings of reduced stress levels were supported by lower Mouse Grimace Scale scores and increased voluntary interaction
• Stress associated with pharmacokinetic studies, caused by procedures such as oral gavage and blood sampling, can be reduced by refined handling and habituation

8. Castronovo & Wesson. 2024. Effects of home cage tunnels on within-cage behaviors of mice with cranial implants. Journal of the American Association for Laboratory Animal Science. DOI: 10.30802/AALAS-JAALAS-22-000087

• Compared to sham craniotomy (control) mice, mice with 1 of 2 types of cranial implants (indwelling cannula or ferrule-type) interacted with tunnels significantly less, indicating that home cage tunnels may not be appropriate enrichment for mice with cranial implants
• Mice were not handled with the tunnels in this study, just observed interacting with them in the cage

9. Swan et al. 2023. Decreased levels of discomfort in repeatedly handled mice during experimental procedures, assessed by facial expressions. Frontiers in Behavioral Neuroscience. DOI: 10.3389/fnbeh.2023.1109886

• Habituated/trained cup-handled mice had lower Mouse Grimace Scale scores after subcutaneous injection and blood collection compared to non-habituated tail-handled mice
• Female mice reached lower Mouse Grimace Scale scores faster than male mice, indicating that females may respond faster to habituation/training

10. Davies et al. 2022. Impact of Refinements to Handling and Restraint Methods in Mice. Animals. DOI: 10.3390/ani12172173

• When assessed by blinded animal care technicians, post-transport mice historically handled via cupping and tunnels had less aversive behaviors to the opening of the shipping box and were easier to handle, compared to mice handled by the tail
• Animals restrained using a modified cupping technique exhibited less aversive restraint-associated behaviors than those restrained using a conventional tail method

11. Novak et al. 2022. Handling method affects measures of anxiety, but not chronic stress in mice. Scientific Reports. DOI: 10.1038/s41598-022-25090-9

• In an experiment using twice weekly brief (2s) handling sessions, tunnel-handled mice housed under standard laboratory conditions demonstrated increased open arm exploration in an elevated plus-maze, and tunnel-handled BALB/c mice demonstrated increased voluntary interaction with the handler’s hand compared to tail-handled mice, even under chronic stress conditions
• When an unpredictable chronic mild stress (UCMS) paradigm was applied for five weeks, there were no significant differences in plasma corticosterone levels or sucrose preference testing between tail-handled and tunnel-handled mice, indicating that tunnel-handling might be compatible with applied chronic stress paradigms
• Tail-handled mice under standard laboratory conditions do not have comparable levels of chronic stress as those created by the UCMS paradigm
  

12. Hull et al. 2022. Effects of non-aversive versus tail-lift handling on breeding productivity in a C57BL/6J mouse colony. PLoS ONE. DOI:10.1371/journal.pone.0263192 

• Tunnel handling increased breeding productivity by 5% (i.e., one extra pup was born and weaned over a 6-month period for each breeding pair) 
• Tunnel handling increased the number of breeding pairs that were able to successfully wean all litters that were produced 
• Tunnel handling decreased the risk of recurrent litter loss by 20% 

NOTE: the results from this study and the projected cost savings as a result of increased breeding productivity were significant enough to persuade a change in the standard of care across the institution (University of Florida)

13. Miller et al. 2022. Welfare assessment, end-point refinement and the effects of non-aversive handling in C57BL/6 mice with Lewis lung cancer. Animals. DOI: 10.3390/ani12010023 

• Non-aversive handling (cupping and tunneling) did not have a significant effect on the data quality or variability in Lew lung carcinoma tumor-bearing mice 

14. Redaelli et al. 2021. Neuroinflammation, body temperature and behavioral changes in CD1 male mice undergoing acute restraint stress: An exploratory study. PLoS ONE. DOI: 10.1371/journal.pone.0259938 

• Tunnel handling and mechanoreceptive handling protected mice from a lack of weight gain seen in tail-handled mice following exposure to restraint and bright light (15min) 
• Mechanoreceptive handling increased the time mice spent in the open arm of an elevated plus maze following exposure to restrain and bright light (15min)

15. Sandgren et al. 2021. Using cage ladders as a handling device reduces aversion and anxiety in laboratory mice, similar to tunnel handling. Journal of Laboratory Animal Science. DOI: 10.23675/sjlas.v47i0.1083 

• Tunnel and ladder handling increased the number of voluntary interactions with handlers 
• Tunnel and ladder handling reduced anxiety, determined by exploration of the elevated plus maze 

16. Clarkson et al. 2020. Negative mood affects the expression of negative but not positive emotions in mice. Proc Biol Sci. DOI:10.1098/rspb.2020.1636 

• Tunnel handling reduced anxiety, as evidenced by increase in the number of voluntary interactions with handlers and time spent in open arms of elevated plus maze 
• Tunnel handling reduced adrenal gland size 
• Tunnel handling increased resilience to negative events 
• Tunnel handling reduced anhedonic behavior and signs of depression, as evidenced by increased sucrose consumption and lick cluster size 

17. Henderson et al. 2020. Benefits of tunnel handling persist after repeated restraint, injection and anaesthesia. Scientific Reports. DOI: 10.1038/s41598-020-71476-y 

• Tunnel handling increases the time spent interacting with handlers and reduces anxiety, as evidenced by increased time spent in the open arms of the elevated plus maze 
• Repeated stressful procedures (scruff restraint, i.p. injections, or anesthesia) do not reverse the benefits of tunnel handling

18. Sensini et al. 2020. The impact of handling technique and handling frequency on laboratory mouse welfare is sex-specific. Sci Rep. DOI:10.1038/s41598-020-74279-3 

• Tunnel handling decreased despair-like behavior in males, but not females, as evidenced by immobilization during forced-swim tests and reduced wellbeing-associated burrowing 
• Tunnel handling increased positive interactions with handlers’ hands (e.g., sniffing, touching, climbing) and reduced negative interactions (defensive burrowing)

19. Ueno et al. 2020. Effects of repetitive gentle handling of male C57BL/6NCrl mice on comparative behavioural test results. Scientific Reports. DOI: 10.1038/s41598-020-60530-4 

• Repeated daily tail handling led to increased body weight compared to mice handled only during cage changes 
• Repeated daily tail handling reduced anxiety, as evidenced by increased time spent in the open arms of the elevated plus maze 
• Repeated daily tail handling improved spatial cognitive function, as evidenced by increased alterations in the Y-maze test

20. Doerning et al. 2019. Assessment of mouse handling techniques during cage changing. JAALAS. DOI:10.30802/AALAS-JAALAS-19-000015 

• Tunnel handling did not increase contamination during cage changes (the surface of the tunnel vs the gloves used to tail handle) 
• Home tunnels did not impair the ability of caretakers to perform cage-side health checks

21. Gouveia et al. 2019. Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice. Scientific Reports. DOI: 10.1038/s41598-019-56860-7 

• Handling for only 2 seconds during cage changes is sufficient to familiarize mice with tunnel handling 
• Brief (10 seconds) but more frequent handling was required to familiarize mice with cupping 
• Tail handling resulted in strong aversion to the handler compared to tunnel handling or cupping 
• Exposure to repeated scruff restraint and subcutaneous injections

22. Mertens et al. 2018. Effect of three different forms of handling on the variation of aggression-associated parameters in individually and group-housed male C57BL/6NCrl mice. PLoS ONE. DOI: 10.1371/journal.pone.0215367 

• Picking up male mice by the tail with forceps, rather than fingers, resulted in stress-induced hyperthermia to a greater degree than tail handling or tunnel handling 
• Forceps-handled mice exhibited increased aggression after cage cleaning compared to tail- or tunnel-handled mice 
• Compared to tail and forceps handling, tunnel handling resulted in reduced anxiety, as evidenced by decreased exit latency and fighting

23. Clarkston et al. 2018. Handling method alters the hedonic value of reward in laboratory mice. Scientific Reports. DOI:10.1038/s41598-018-20716-3 

• Tunnel handling reduced depressive-like behaviors, as evidenced by increased sucrose consumption and lick cluster sizes

24. Nakamura & Suzuki. 2018. Tunnel use facilitates handling of ICR mice and decreases experimental variation. Journal of Veterinary Medical Science. DOI: 10.1292/jvms.18-0044 

• After 1 week of handling and 1 week of oral gavage, tunnel-handled mice exhibited increased voluntary interactions with the experimenters compared to tail-handled mice 
• Tunnel-handled mice were easier to handle and urinated and defecated less during handling, as measured by a “rating scale for wildness” 
• Tunnel handling increased exploration in the open field test and the elevated plus maze compared to tail handling, but did not increase %time spent in the open arms of the elevated plus maze 
• Tunnel handling reduced the variation for the data collected behavioral tests (open field test and elevated plus maze) after IP administration of saline or diazepam

25. Roughan & Sevenoaks. 2018. Welfare and scientific considerations of tattooing and ear-tagging for mouse identification. Journal of the American Association for Laboratory Animal Science. DOI: 10.30802/AALAS-JAALAS-18-000057 

• Tunnel handling increased voluntary interaction with handlers compared to tail handling, even after restraint, tattooing, or ear-tagging 
• Tunnel handling resulted in reduced Mouse Grimace Scale scores after acclimation to handling and following restraint, ear-tagging, or tattooing
  

26. Gouveia & Hurst. 2017. Optimising reliability of mouse performance in behavioral testing: the major role of non-aversive handling. Scientific Reports. DOI: 10.1038/srep44999

• Tunnel-handled female mice showed increased willingness to explore and investigate test stimuli (urine samples collected from male mice), regardless of familiarization or stimulus location, compared to tail-handled mice 
• Tunnel- and cup-handled mice overall exhibited less caution and increased exploratory behavior compared to tail-handled mice during behavioral testing (habituation-dishabituation paradigm) 
  

27. Wilde et al. 2017. Tail-cuff technique and its influence on central blood pressure in the mouse. Journal of the American Heart Association. DOI: 10.1161/JAHA.116.005204 

• Handling method (tail, tail-cup, or tunnel) did not elicit differences in blood pressure, heart rate, or body temperature before, during, or after restraint for tail-cuff plethysmography 
• The authors concluded that restraint induces sustained, high levels of stress in mice and that the findings of this study should be considered in designing studies that involve the tail-cuff technique
  

28. Ono et al. 2016. Does the routine handling affect the phenotype of disease model mice? Japanese Journal of Veterinary Research. DOI: 10.14943/jjvr.64.4.265 

• Tail handling resulted in increased severity of glomerulus lesions compared to control (non-handled) mice in a model of ICR-derived glomerulonephritis 
• Tail handling resulted in increased plasma corticosterone levels in C57BL/6 mice (but not BALB/c mice) compared to controls; 
• Tunnel handling resulted in increased plasma corticosterone levels in BALB/c mice (but not C57BL/6 mice) compared to controls 
• The authors noted that it took much longer for BALB/c mice to enter the tube voluntarily (5min) compared to C57BL/6 mice (10-15sec), indicating potential strain differences in handling-induced stress 
  

29. Ghosal et al. 2015. Mouse handling limits the impact of stress on metabolic endpoints. Physiology & Behavior. DOI:10.1016/j.physbeh.2015.06.021

• Cupping reduced anxiety-like behaviors and blood glucose levels 
• Cupping resulted in improved glucose tolerance in mice fed a high fat diet 
• Cupping + massage resulted in lower glucose levels following an overnight fast 
• Cupping + massage reduced anxiety-like behaviors and decreased plasma corticosterone levels
  

30. Miller & Leach. 2015. The effect of handling method on the mouse grimace scale in two strains of laboratory mice. Laboratory Animals. DOI: 10.1177/0023677215622144 

• Handling method alone did not result in significantly different Mouse Grimace Scale scores (tail vs tunnel handling), demonstrating that handling method is not a confounding factor for determining a baseline score 
  

31. Novak et al. 2015. An exploration based cognitive bias test for mice: effects of handling method and stereotypic behaviour. PLoS ONE. DOI: 10.1371/journal.pone.0130718 

• No difference was found between handling methods (tail handling vs cupping) in the level of exploration of mice in the radial arm maze (arms were positively or negatively cued); a notable caveat: mice were housed two to a cage such that one mouse was tail handled while the other was handled by cupping, potentially confounding the results if stress is communicated amongst cage-mates (I.e. the mouse handled by cupping may have experienced stress unrelated to the handling method as a result of being housed with a stressed, tail-handled cage-mate)
  

32. Gouveia & Hurst. 2013. Reducing mouse anxiety during handling: Effect of experience with handling tunnels. PLoS ONE. DOI: 10.1371/journal.pone.0066401 

• Tunnel handling reduced anxiety in both C57BL/6 and CD-1 mice with both home cage tunnels and shared tunnels, regardless of prior familiarity, as evidenced by increased willingness to approach the handler and behavior in the elevated plus maze) 
• Home cage tunnels may be more beneficial for anxious strains, as C57BL/6 mice showed faster habituation to handling with a home cage tunnel compared to shared tunnel (evidenced by increased voluntary interaction with the tunnel)
  

33. Hurst & West 2010. Taming anxiety in laboratory mice. Nat Methods. DOI:10.1038/nmeth.1500 

• Tunnel handling & cupping were found to reduce stress/anxiety, as evidenced by approach behavior with handlers, urination/defecation during handling, and the elevated plus maze, for multiple strains & during both light and dark phases. 
• Tunnel handling & cupping are compatible with scruff restraint & tail lifting. 
• Tunnel handling and cupping were effective regardless of handler