Relieving physical fatigue function testing is the core efficacy evaluation required for health supplements and health foods that carry an anti-fatigue claim, particularly under the Chinese health food registration framework. This technical guide explains the sample requirements, key testing items, animal-based testing methods, judgment criteria, and typical applications of anti-fatigue function testing performed by accredited third-party laboratories in compliance with the relevant national standards and evaluation methods.
Technical Overview
Health supplements and health foods that are marketed with the claim of helping to relieve physical fatigue must provide scientific evidence demonstrating that the product genuinely possesses this function. In China, this claim is evaluated according to the official function testing and evaluation methods for health foods issued by the national market regulation and health authorities, most recently codified in the 2023 edition of the Function Testing and Evaluation Methods for Health Foods, which superseded the earlier 2003 Technical Specification for Testing and Assessment of Health Food.
The evaluation is an animal-based experiment conducted on healthy male mice. Animals receive the test sample by oral gavage for a continuous administration period of at least 30 days, after which four core endpoints are measured: weight-loaded swimming endurance time, blood lactic acid levels, serum urea concentration, and hepatic glycogen content. These endpoints collectively reflect exercise capacity, metabolic acid accumulation, protein catabolism, and energy reserve status.
A positive conclusion requires a statistically significant improvement in the weight-loaded swimming test together with a favorable result in at least one of the three biochemical indicators. In parallel, registration dossiers are supported by safety and quality testing performed according to national standard series such as GB 15193 for toxicological assessment, GB 4789 for microbiological examination, GB 5009 for physicochemical analysis, and GB 14925 for laboratory animal care and housing. Reliable testing is performed by accredited third-party laboratories operating under an ISO/IEC 17025 quality management system and holding valid laboratory animal use permits.
Testing Samples
Finished Health Food Products
Testing is typically performed on the finished product in its final commercial formulation, ensuring that the evaluated sample is representative of the product that will reach consumers. Common dosage forms include:
- Tablets, chewable tablets, and effervescent tablets
- Hard capsules and soft capsules
- Granules and powdered mixes
- Oral liquids, concentrated drinks, and beverage formulations
- Gummies, confectionery-based supplements, and other novel delivery forms
Functional Raw Materials and Extracts
Ingredient suppliers and product developers may also submit functional raw materials for evaluation prior to formulation, including ginseng root extract, American ginseng extract, Cordyceps militaris mycelium, taurine, branched-chain amino acids, creatine monohydrate, B-vitamin complexes, oyster or fish collagen peptides, and traditional herbal composite extracts.
Sample Submission Requirements
Samples should originate from a representative production batch with confirmed homogeneity and stability. The quantity submitted must cover the entire dosing period for all animal groups and all four endpoint tests, with an additional reserve. The human recommended daily intake stated on the product label serves as the basis for converting to animal dose levels, so complete formula and dosage information must accompany the submission. Liquid and paste samples are gavaged directly or after dilution, while solid samples are ground, suspended, or dissolved in a suitable vehicle such as purified water or a low-concentration thickening agent.
Testing Items
The core efficacy endpoints of the relieving physical fatigue function test, together with the supporting observations, are listed below:
- Weight-loaded swimming endurance time, the primary indicator of exercise capacity
- Blood lactic acid concentration at defined time points and the calculated area under the blood lactic acid curve
- Serum urea concentration following a standardized swimming session
- Hepatic glycogen content determined from liver tissue
- Body weight, food intake, water intake, and general health observations throughout the administration period
- Supporting analyses where required, such as active marker quantification, microbial limits, and contaminant testing in accordance with GB 4789 and GB 5009 series standards
Testing Methods
Animal Study Design
Healthy adult male mice, generally weighing 18 to 22 grams at study initiation, are acclimated for several days and housed under controlled environmental conditions in accordance with GB 14925. Animals are randomly assigned to a negative control group and at least three dose groups, typically set at multiples of the human recommended daily intake, for example five-fold, ten-fold, and thirty-fold equivalents converted on a body-weight basis. The test sample is administered daily by oral gavage for at least 30 consecutive days, with gavage volumes kept within accepted animal welfare limits. All procedures are reviewed and approved by the institutional animal ethics committee.
Weight-Loaded Swimming Test
Following the final dose, mice undergo a swimming endurance test in which a lead wire or equivalent load equal to a fixed percentage of body weight, commonly five percent, is attached at the base of the tail. Mice swim individually in water tanks with a controlled water temperature, typically maintained between 25 and 30 degrees Celsius, and sufficient depth to prevent passive support. The swimming time is recorded until exhaustion, defined as the point at which the animal fails to surface within a prescribed observation window. A significantly prolonged swimming time in dose groups compared with the negative control indicates enhanced exercise capacity.
Blood Lactic Acid Determination
Blood lactic acid is measured at rest, immediately after a standardized ten-minute unloaded swimming session, and again after a defined recovery period, commonly twenty minutes. The area under the blood lactic acid curve is calculated by the trapezoidal method across the three sampling points. Lactic acid is quantified using an enzymatic lactate dehydrogenase-based method. A reduced area under the curve indicates improved clearance or attenuated accumulation of metabolic acid, which supports an anti-fatigue effect.
Serum Urea Determination
Serum urea is measured after a standardized prolonged unloaded swimming session, commonly ninety minutes, followed by a defined rest period before blood collection. Urea concentration is determined using a urease-based enzymatic assay. Because urea reflects the degree of protein catabolism under exercise stress, a significantly lower serum urea level in treated animals compared with controls indicates reduced metabolic stress and more efficient energy utilization.
Hepatic Glycogen Determination
Hepatic glycogen is quantified from liver tissue collected under standardized exercise conditions using the anthrone colorimetric method. Glycogen is the principal carbohydrate energy reserve mobilized during sustained physical activity, and a significantly elevated hepatic glycogen content in treated animals indicates an improved energy reserve status, supporting endurance enhancement.
Data Processing and Judgment Criteria
All results are analyzed with appropriate statistical methods, typically analysis of variance followed by pairwise comparisons, with a significance level of five percent. Body weight and food intake data are evaluated to exclude confounding effects unrelated to genuine anti-fatigue activity, such as reduced appetite or altered growth. The test sample is judged to possess a relieving physical fatigue function when the weight-loaded swimming time is significantly prolonged and, in addition, at least one of the three biochemical indicators, namely blood lactic acid, serum urea, or hepatic glycogen, shows a statistically significant favorable result. If the swimming endurance endpoint is negative, a positive function claim cannot be supported regardless of the biochemical results.
Applications
Relieving physical fatigue function testing serves multiple purposes across the product lifecycle of health supplements:
- Registration support for health food approval dossiers, providing the efficacy data package required by regulatory authorities for function claim approval
- Research and development screening, allowing formulators to compare candidate formulas, optimize dose levels, and select superior ingredient combinations
- Claim substantiation for marketing and e-commerce documentation, supplying objective laboratory evidence behind anti-fatigue advertising statements
- Export compliance support, where products targeting multiple markets require efficacy data aligned with the requirements of the destination jurisdiction
- Quality control and stability studies, confirming that the anti-fatigue effect is maintained across production batches and throughout the intended shelf life
- Competitive benchmarking, enabling objective comparison of a product against market reference formulations
FAQ
How long does the complete test take?
A standard study, including sample preparation, animal acclimation, 30 days of continuous administration, endpoint testing, statistical analysis, and report issuance, typically requires approximately six to eight weeks. Additional time should be reserved if supporting safety or quality tests are performed in parallel.
How are animal dose levels determined?
Dose levels are derived from the human recommended daily intake stated on the product label. The daily intake is converted to a mouse-equivalent dose on a body-weight basis and then set at defined multiples, commonly five-fold, ten-fold, and thirty-fold, to establish a low, medium, and high dose group.
What result is required to claim the anti-fatigue function?
The weight-loaded swimming test must show a statistically significant increase in endurance time, and at least one of the three biochemical indicators, blood lactic acid, serum urea, or hepatic glycogen, must also show a significant favorable improvement compared with the negative control group.
Are human clinical trials accepted as an alternative?
Under the current evaluation framework for this specific function claim, animal testing remains the recognized basis for efficacy evaluation. However, human studies using endpoints such as perceived exertion ratings, heart rate recovery, and exercise performance can provide valuable complementary evidence for marketing substantiation in markets where such claims are regulated differently.
What qualifications should the testing laboratory hold?
Testing should be entrusted to accredited third-party laboratories that operate an ISO/IEC 17025 compliant quality system, hold a valid laboratory animal use license, maintain animal facilities meeting GB 14925 requirements, and have documented experience with health food function evaluation methods.