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"HOW TO" GUIDE FOR TESTING PROTEIN PRODUCTS: A Resource for Retailers, Industry and Consum

You see it all the time; protein-based products being tested by on-line shopping destinations, consumer advocate entities and even self-anointed fitness gurus. The goal of most is to validate, or even to invalidate, nutrient levels claimed on the Nutrition Facts or Supplement Facts panel as well as elsewhere on the label. Protein is always in the crosshairs of assessment since it is the main ingredient that drives the perceived value of the product. In fact, some savvy protein product shoppers will “do the math”, factoring the price tag against protein content (e.g $/lb protein), when deciding which product to purchase.

In addition to protein, testing typically includes calories, total and type of fats, cholesterol, carbohydrate, sugar, fiber, sodium and potassium. In fact, discrepancies between the protein levels listed on product labels and test results have fueled numerous “Amino Spiking” lawsuits as well as other nutrients over the years (see Protein Supplements: Don’t get Fooled). Despite the unquestionable usefulness of product testing to foster consumer confidence, one thing that is clear to is that the nuances of laboratory testing for nutritional composition are not always fully understood by the tester. This can lead to misunderstandings and inaccurate reporting of nutrient levels related nutrient claims.

On the other hand, testing nuances has opened the door for some trickery by knowledgeable formulators resulting in misleading labeling of some nutrients including protein. In this article will overview some of the most important aspects when it comes to understanding protein product labels as well as testing and evaluating test results of protein-based products. In addition, we will include some additional detail as to the federal regulations related to product testing.

Food and Supplements: What’s the Difference?

Food and supplements are similar in some ways and different in others, but both fall under the guidance of the Food and Drug Administration (FDA). First, food and supplement ingredients must be found in nature and in some manner part of the human diet. Even if a nutrient is synthesized, its identity must still match the nutrient found in mother nature’s planet earth buffet. From there, the key major differences between foods and supplements include:

  • Intention of use – Is it meant to nourish the body with energy and macronutrients and consists of a major meal component? Or, is it meant to add additional nutrients but not necessarily replace a food or substitute for a meal?

  • Product Form – Is it a beverage, bar or other form that is associated more with food? Or is it a pill, chew, shot, etc., more associated with a supplement to the diet?

  • Serving size vs RACC – How similar is a serving or portion size to what’s commonly consumed for foods? For instance, is a serving of a liquid 8 to 12oz or is the product a nutrition bar? Food serving amounts are referred to as RACC (Reference Amount Customarily Consumed). Because these are more food-like, they require a Nutrition Facts panel.

  • Product Composition – Does the product consist of Individual or combined nutrients at specific levels vs commodity ingredient(s) as well as classification of nutrients. For the latter, foods need to include only ingredients that are classified as Generally Recognized as Safe (GRAS) by the FDA.

Basically, a manufactured food needs to provide significant nourishment including meaningful levels of calories and/or macronutrients namely protein, carbohydrate, fat and water. Plus, a manufactured food must be a form and amount typically consumed. That means that a beverage with a serving size of 8-12oz is more food-like. Plus, if the directions for a nutritional powder or gel states to add water yielding a consumable beverage with a serving size of 8-12oz, then it too would be food-like. On the other hand, a 2 to 5 oz energy shot would be more supplement like, especially if the primary ingredients are caffeine flanked by vitamins, etc.

Compliance with FDA Guidance for Labeling

Labeling accuracy is important, and any claims made regarding nutrient levels must meet certain guidelines. In the United States the FDA classifies nutrients that are declared on Nutrient Facts and Supplement Facts as either Class I or II as follows:

  • Class I Nutrients - Nutrients that are specifically added to food (e.g. fortified food) to increase its nutritional value or active nutrients formulated as part of a dietary supplement. These nutrients include added vitamins, minerals and fiber as well as nutraceutical nutrients such as amino acids (e.g. BCAAs, taurine, citrulline, caffeine, plant extracts, etc.). Class I nutrients must be present at 100% or more of the value declared on the label all the way to the end of a product’s expiration date. Class I nutrients would include vitamins and minerals added to a breakfast cereal as well as nutrients formulated into a multivitamin/mineral supplement. In addition, if a specific nutrient is called out in the Statement of Identity on the front of the package (e.g. Protein Powder or Protein Bar) then the protein listed in the Nutrition Facts panel is considered a Class I nutrient. The same is true if a product makes a nutrient content claim somewhere on the package/label (e.g. “10 grams of Protein”) in a food not commonly assumed to deliver that nutrient in significant amounts, then in this case protein could be classified as a Class I nutrient as well.

  • Class II Nutrients – Nutrients that are naturally occurring in an intact food (e.g. fruits, vegetables, oatmeal) or recipe foods (e.g. baked good, peanut butter) but can vary in concentration for reasons that cannot be controlled or predicted easily. Examples would be vitamin C in picked oranges or calcium in whole milk. Class II nutrients must be present at 80% or more of the value declared on the label.

  • Third Group Nutrients - In addition, specific nutrients of health-related interest are grouped together as “third Group” which cannot exceed 120% of label claim. Third group nutrients include calories, sugars, total fat, saturated fat, cholesterol, and sodium.

Nutrition Facts Testing

Many labs offer to bundle total Nutrition Facts composition since the components are set by the FDA as part of the NLEA (Nutrition Labeling and Education Act). Some of this information has changed in 2020 and the Nutrition Facts must include: Calories, Total Fat, Saturated Fat, Total Carbohydrate, Fiber, Sugar, Cholesterol, Sodium, Iron, Calcium and Potassium and Vitamin D. In addition, “Added Sugars” takes its place below Sugar in the Nutrition Facts, to help consumers understand levels of naturally occurring sugars from sugars added to recipes. The FDA provides a Comparison of Old and New Nutrition Facts.

Ordering bundled Nutrition Facts testing can make it easier and less expensive; however, there are some notable considerations as some of the information you need might require additional testing to be more accurate on the label. Examples are additional analysis for certain added fibers that are not typically captured in general fiber testing methods. Additionally, sugar alcohols and numerous vitamins and minerals must be requested if they are not part of the Nutrition Facts nutrient listings.


Calories are an easy assessment for analytical labs because in most cases total calories are a mathematical sum and not a direct analytical measurement. Food calories are derived from one of four nutrient families (carbohydrate, fat, protein and alcohol) so calories can be estimated accordingly simply by adding the calories from the four main energy nutrient classes as follows:

Total Calories = Carbohydrate Calories* + Protein Calories + Fat Calories + Alcohol Calories

*Accuracy depends on method used and ingredients

However, the accuracy in estimating calories will in turn depend on the accuracy in assessing the other nutrients. For instance, an overestimation in carbohydrate could falsely overestimate calories. Meanwhile, certain nutrients need to have the appropriate calorie factor applied such as sugar alcohols, certain fiber types, organic acids or “acids” (e.g. malic acid, citric acid).


Protein is the most interesting nutrient class, especially when it comes to sport protein products. Proteins are molecules comprised of amino acids linked together by peptide bonds. Protein is viewed as the most highly valued and recognizable macronutrient in sports nutrition, however not all food protein isolates and concentrates (e.g. whey, soy, rice) have the same nitrogen density, bioavailability and potency…. as well as price tag.

There are a few main FDA rules for protein-related labeling which include:

  • Protein content may be calculated on the basis of the factor 6.25 times the nitrogen content of the food as determined by the appropriate method of analysis as given in the "Official Methods of Analysis of the AOAC International," except when official AOAC procedures described in this paragraph (c)(7) require a specific factor other than 6.25, that specific factor shall be used (21CFR101.9(c)(7)). This conversion factor is based on the long-standing knowledge that high-protein foods commonly consumed (e.g. meats, eggs, fish) are about 16% nitrogen. However, different proteins have unique nitrogen concentrations. This leaves opportunity for more specific factors to be applied based on single or specific recipes of protein sources as described below.

  • Supplements containing only amino acids cannot declare protein on the label. (21CFR 101.36(b)(2)(i) - This rule is important because the amino acids should be formulated in for unique properties outside providing a building block for making protein in the body. That’s because many amino acids: 1) serve as precursors for other important, non-protein molecules in the body, 2) are not used as building block amino acids to make protein in the body. For instance, Beta-alanine is used to make the muscle buffer carnosine, taurine is not used as a building block amino acid either.

  • Protein Daily Value (DV) is calculated by correcting the actual amount of protein in grams per serving by multiplying the amount by its amino acid score corrected for protein digestibility, dividing by 50 grams, and converting to percent (21CFR 101.9(c)(7)(ii))

Issues and manipulations with both FDA labeling guidelines are addressed below. These should be considered during any evaluation of protein-based supplements, which by and large make up most of the testing in the sport nutrition industry done by consumers, websites and professional organizations.

Total Protein Testing

Total protein is assessed in a very simple and crude manner. Here is how it works. Since, carbohydrate, fat, water and the major minerals do not contain nitrogen, the nitrogen in a food is assumed to be from protein. Thus, by determining total nitrogen, a factor can be used to derive total protein. For instance, the FDA specifies that grams of nitrogen be multiplied by 6.25 to estimate grams of protein based on the general knowledge that protein in the high-protein foods commonly consumed (e.g. meats, eggs, fish) is about 16%. However, the conversion factor for milk proteins is 6.38 as the nitrogen content is a little less concentrated and the conversion factor for vegetable proteins is lower (e.g. 5.70) because their nitrogen concentration is higher (see table). This means that a milk protein-based product containing only whey and/or casein and/or milk protein concentrate should use a conversion factor of 6.38 and not 6.25.

Meanwhile a blended protein containing a mixture of milk protein and/or egg and vegetable proteins needs to use a conversion factor reflective of the protein contribution from each ingredient or at the least revert to 6.25 if the blend is somewhat balanced. Meanwhile, a protein product that is mostly or exclusively vegetable protein should use the correct nitrogen factor for the sources. The primary reason for the difference in conversion factors is the proportion of amino acids in the proteins. Proteins that have a higher content of amino acids such as arginine, with its four atoms of nitrogen, should have a higher nitrogen concentration, otherwise it will overestimate protein content.

Amino Spiking Investigation

One of the biggest issues with protein labelling in the sport nutrition market is that based on the limited guidance for protein labeling, other sources of non-protein nitrogen sources added to a formulation could also be counted towards protein since the general testing for nitrogen content does not discriminate. For instance, if creatine or betaine are added to the formulation, they will contribute nitrogen, which in turn will be pooled with protein nitrogen to estimate total protein. While creatine and carnitine are derived from amino acids, they are not converted back to amino acids for protein manufacturing in the human body.

Moreover, if amino acids are also added to formula, they too will add nitrogen and are often counted towards protein. That means that some products containing three grams of the amino acid taurine, which is not used to make protein in the body, will be counted as protein on the label. The same is true for glycine, which is a nonessential amino acid, and not really a strategic addition to a protein product positioned to build muscle. Glutamine is another amino acid that has been added to protein products to increase the nitrogen content. Here again, the nitrogen from this amino acid must be factored out prior to the application of a nitrogen conversion factor to estimate protein.

The primary reason for this practice is to lower costs of the formulation. In general, nitrogen containing ingredients such as creatine, glycine, taurine, etc. are less expensive than intact protein when you look at as the $/g protein that would be derived inappropriately.

Amino Acid Profile Testing

The important thing to keep in mind is that simple nitrogen testing protocol does not discriminate between protein types nor will it tease out non-protein nitrogen. This means that any additional amino acids and other nitrogen containing molecule could be counted as protein on the label. To drill down further, one could have an amino acid profile performed which would identify how much of specific amino acids are in a serving of a product. Furthermore, a lab will be able to perform a test in which they can provide amino acid levels bound in proteins versus free amino acids, which would largely be added to a formula. Also, be sure that the amino acid profile includes taurine and hydroxyglycine if taurine or collagen are listed as an ingredient on the label. This is important for many consumers that are targeting a protein supplement toward muscle protein building as neither will be used to make muscle protein or protein anywhere else in the body.

Calories from Amino Acids

Brands of conventional foods and dietary supplements must adhere to the FDA guidelines in labeling Total Calories in any such Dietary Supplement and Food product. Even though amino acids formulated as part of supplements such as BCAA (Branched Chain Amino Acid) or EAA (Essential Amino Acid) products and pre-workouts cannot be listed as protein (21CFR 101.36(b)(2)(i)) on the Supplement Facts panel, their calories still need to be accounted for per 21 CFR 101.9(c). Amino acid-based products with a significant caloric value (e.g. > 5 calories/serving) should label Total Calories and consider these calories combined with other often missed calorie contributors such as organic acids (e.g. malic acid, citric acid). On the other hand, if the total caloric level is <5 calories/serving they do not have to be accounted in the Nutrition or Supplement Facts panel (21 CFR 101.9(c)(1)).

Calorie-containing amino acids would include any of the twenty protein building block amino acids (or proteogenic amino acids) as well as citrulline. Calculation of caloric value is equal to grams of amino acid(s) x caloric value/gram. In general, 4 kcal/gram is the default factor. Meanwhile amino acids such as beta alanine and taurine have nominal caloric value. Similarly, amino acid derivatives such as carnitine, glutathione and creatine cannot be converted back to amino acids and should not be counted as calories.


Total carbohydrates are typically estimated based on a very crude “subtraction method”, much like you learned in grade school. Here is how it works: Once moisture, protein, fat, cholesterol and ash (minerals) is accounted for by independent testing methods (Appendix A), what is left over is assumed to be carbohydrate. That’s it! That means any inaccuracies in the “subtractables” will affect the accuracy of the estimate of carbohydrate.

Total Carbohydrate = 100% - (Protein + Ash (Mineral) + Moisture (Water) + Fats + Cholesterol)

The subtraction method tends to be more precise with intact foods such as fruits, vegetables, meats, eggs, milk, etc. However, for more complex recipe foods this method might overestimate total carbohydrate when certain sweeteners, colors, flavors, stabilizers (gums), acids, etc. are used. This often leads to confusion when the known level of sugars, starch and fiber don’t add up to the total carbohydrate on the lab report. In addition, the accuracy of calculated Total Carbohydrate is in turn affected by the accuracy of tested nutrients such as protein, moisture, minerals (ash), etc.

Low Carbohydrate Foods

For products that are formulated to be lower in digestible carbohydrates (e.g. sugars and starches) it is often helpful to have additional testing done for digestible carbohydrates to yield a more accurate estimation of Total Carbohydrate. This testing involves additional enzymes to breakdown starch to simple sugars which are then quantified through testing and compared to levels before enzyme treatment. The difference in sugar content (e.g. glucose) before and after enzyme treatment would be a more accurate estimation of starch content. (See Appendix A)

In addition, some ingredients that improve the sensory properties of a recipe, might contain maltodextrin as a “carrier”. This would contribute to the final starch count albeit small. Lastly, many low carb foods are formulated with sugar alcohols and functional fibers and resistant starches which are discussed below.


Sugars include glucose, fructose and galactose, which are all monosaccharides and serve as building blocks for disaccharides found in foods such as maltose, lactose (milk sugar) and sucrose (table sugar). Different foods can have different proportions of various sugars, so testing for sugars should be food specific (see Table 1). Protein products should include testing for simple sugars to more accurately estimate sugar as well as Total Carbohydrate. For instance, if a milk-protein based formulation does not include starch (e.g. maltodextrin) or other non-fiber carbohydrate, then the amount of sugar, which is primarily lactose, can be used to more accurately label Total Carbohydrate. Egg protein sources are extremely low in sugar and carbohydrate too. On the other hand, some vegetable protein ingredients can have some naturally occurring starch.

Starches like maltodextrin will have some sugar as glucose and maltose, although the content is typically low (3 to 5%). Maltodextrin might be used as a carbohydrate energy source for recovery products and gainers. Thus, adding 50 grams of maltodextrin to a gainer product could add 1½ to 2 ½ grams of sugar and it is often used as a carrier for flavors and other ingredients. In addition, partially digested starches will have some sugar content and needs to be accounted for on the label. Thus, starch-based ingredients will contribute to Total Carbohydrate as well as Sugar on the Nutrition/Supplement Facts.

Added Sugar

Previous Nutrition Facts do not distinguish between naturally occurring sugars in foods and sugars added as sweeteners. However, as of 2020, the latter will need to be declared on the Nutrition Facts panel under Total Carbohydrate and Total Sugar as “Includes Xg of Added Sugar”. Based on methods, basic testing for sugar would not directly distinguish between naturally occurring sugars and those that are potentially added for sweetening/sensory purposes. However, there are some simple solutions. For instance, the sugar in milk proteins (e.g. whey, casein, milk protein) will be mostly lactose, which is half galactose. So, determination and doubling of galactose will estimate total lactose and thus the sugar naturally occurring in milk protein ingredients.

Corn syrup is sometimes called glucose syrup and typically is about 30-35% glucose. Meanwhile, other starch providers can be used to yield a glucose syrup including tapioca, wheat and potato. Fructose is the principle monosaccharide primarily found in sucrose, fruit ingredients and syrups. Sucrose is half fructose while most common high fructose corn syrup (HFCS) is 42-55% fructose with the lower end typically used for recipes and baked goods and the higher end of the range for beverages.

Allulose is a more recent consideration to sugar claims. Allulose is a form of fructose that is nearly identical in molecular design to fructose. That allows allulose to have some of the sweetness as fructose, just not as much. However, it’s different enough that allulose is not processed for energy like fructose. Based on these characteristics, allulose must be listed as carbohydrate on Nutrition and Supplement Facts labels, but doesn’t need to be counted as calories or added sugar.

Fruit juice concentrates that are added to a recipe to sweeten the product must also be factored into added sugar declarations. Fruit juices have more fructose than other sugars and thus are generally sweeter than sucrose (table sugar). Meanwhile, juice concentrates that are to be reconstituted to original water content would be exempt.

Sugar Alcohols

Sugar alcohols became popular in sugar-free gums and candies when it was determined that they can be used to sweeten foods such as candies but not promote dental cavities like simple sugars. Today, sugar alcohols such as erythritol, maltitiol, xylitol, and glycerol are used to sweeten many foods and either in place of sugar or in combination with sugar to reduce the amount used. The listing of Sugar Alcohols under Total Carbohydrate on the Nutrition Facts panel is voluntary unless a claim is made on the package related to sugar and/or sugar alcohol.

Among the biggest concerns for sugar alcohols is the risk of discomfort in the digestive tract and thus some people avoid them all together or minimize their intake. Erythritol seems to be the most tolerable of the sugar alcohols. When it comes to labeling accuracy, these ingredients should be counted towards Total Carbohydrate, but their calorie contribution should be assessed according to the acceptable food energy they provide (Table 2).


Fiber analysis is based on general, crude methods established long ago. While some greater accuracy can be gained by certain classic assessment methods for fiber, the fiber content of foods in their natural state or produced via traditional processing or recipes is accurate. However, more modern food processing and availability of functional fibers (e.g. inulin from chicory root) can go undetected by traditional testing and must be tested for specifically to accurately determine total and individual fiber content. Therefore, testing for fiber content of a food/supplement should apply the appropriate testing protocols.

More recently, some food companies have counted certain carbohydrate ingredients as fiber, although they are not. This was the case for IMO or isomalto-oligosaccharide. The most seminal definition aspect of fiber is that it generally remains undigested by human digestive enzymes. Based on this there should not be a significant glycemic response in the hours that follow consumption, but there should be some degree of bacterial fermentation in the colon and the creation of gases like H2 (Hydrogen Gas) which is exhaled. Thus, in addition to lab testing, some fibers can be put to the test via glycemic response and hydrogen breath testing.


Approved Fibers for Labels

According to the last FDA Guidance of fibers for nutrition labeling purposes, declaration can include one or more of the eight reviewed and approved isolated or synthetic non-digestible carbohydrates (Table 3). When present and included in the amount of dietary fiber declared on the Nutrition Facts label.

Total Fat, Saturated and Trans Fat, Other Fats and Cholesterol

Testing protocols for fats and cholesterol are basic and without major considerations. Saturated fat and trans fat require mandatory labeling information and should be part of routine Nutrition Facts testing packages. On the other hand, monounsaturated and polyunsaturated fats are voluntary listings and often must be specifically requested as part of the report. The additional testing for the monounsaturated and polyunsaturated testing would be helpful for products that are more aligned with heart health.

Calories from fat is determined by multiplying Fat by 9 and then rounding rules are applied to simplify labeling information for the consumer. Listing Calories from Fat is no longer a mandatory label requirement as of 2020.

Organic acids tend to fall into Total Carbohydrate estimates based on the mathematical subtraction method above. Meanwhile, for some dietary supplement, malate can be found combined with amino acids (e.g. citrulline malate). In another dietary ingredient creatine citrate malate would need to count the calories from malate as well as citrate.

Vitamins and Minerals

Only vitamin D and four minerals (sodium, calcium, potassium and iron) are mandatory listings for a Nutrition Facts panel, but not for Supplement Facts unless in significant amounts. The listing of other vitamins and minerals are voluntary on either Nutrition and Supplement Facts panels unless they are fortified in the recipe. Vitamin A testing presents a little challenge since it would include preformed vitamin A (e.g. retinol) as well as carotenoids (e.g. beta carotene) with conversion potential.

  • Total Minerals (Ash) - Total mineral content is often referred to as “ash” based on the incineration method applied. This process is straight forward as the assumption is that organic material will be negated and only inorganic materials consisting of minerals would remain. Once total ash is determined it is used to help determine total carbohydrate as described above.

Water (Moisture)

Water is not part of a Nutrition Fact panel although it is routinely assessed by labs. One reason is that it clarifies quality and potential change over time. In addition, it used to estimate total carbohydrate like minerals (ash). Water (moisture) content is often determined by weighing a food item before and after drying in an oven-like apparatus. The difference between the “as-is” weight and the “dry weight” is water or moisture content.

Organic Acids

Organic acids include malic acid, citric acid, tartaric acid, etc., but not phosphoric acid. These ingredients are often added to increase the acidity of a formulation as well as to enhance a fruity flavor. However, these nutrients don’t easily fall into the classification for other energy providers, such as carbohydrates, fat, protein and alcohol, but they do provide energy. Biologically these nutrients are intermediates of the energy pathways that drive energy production in cells. So, their energy content needs to be understood and addressed. For instance, the caloric level of malic acid (malate) is 2.4 cal/gram.

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