When symptoms are limited to gastrointestinal effects, there may be an overlap with wheat allergy, irritable bowel syndrome (IBS), and (less likely) intolerance to FODMAPs.Proposed criteria for a diagnosis of NCGS suggest an improvement of gastrointestinal symptoms and extra-intestinal manifestations higher than 30% with a gluten-free diet (GFD), assessed through a rating scale, is needed to make a clinical diagnosis of NCGS. To exclude a placebo effect, a double-blind placebo-controlled gluten challenge is a useful tool, although it is expensive and complicated for routine clinical use, and so is usually performed in research studies.These suggestions were incorporated in the Salerno expert consensus on diagnostic criteria for NCGS. These recommend assessment of the response to a 6-week trial of a gluten-free diet using a defined rating scale (Step 1), followed by a double-blind, placebo-controlled challenge of gluten (or placebo) for a week of each (Step 2). A variation of greater than 30% in the main symptoms when challenged by gluten or placebo is needed for a positive result. Further research on possible biomarkers was also identified. Differential diagnosis
Examinations evaluating celiac disease and wheat allergy should be performed before patients remove gluten from their diet. It is critical to make a clear distinction between celiac disease and NCGS. Celiac disease
The main goal in diagnosing NCGS is to exclude celiac disease.
Gluten
It was hypothesized that gluten, as occurs in celiac disease, is the cause of NCGS. In addition to its ability to elicit abnormal responses of the immune system, in vitro studies on cell cultures showed that gluten is cytotoxic and causes direct intestinal damage. Gluten and gliadin promote cell apoptosis (a form of programmed cell death) and reduce the synthesis of nucleic acids (DNA and RNA) and proteins, leading to a reduction in the viability of cells. Gluten alters cellular morphology and motility, cytoskeleton organization, oxidative balance and intercellular contact (tight junction proteins). Other proteins
Some people may have a reaction to other proteins (α-amylase/trypsin inhibitors [ATIs]) present in gluten-containing cereals that are able to inhibit amylase and trypsin. They have been identified as the possible activator of the innate immune system in celiac disease and NCGS.ATIs are part of the plants natural defence against insects and may  cause toll-like receptor 4 (TLR4)-mediated intestinal inflammation in humans. These TLR4-stimulating activities of ATIs are limited to gluten-containing cereals (wheat, rye, barley, and derivatives) and may induce innate immunity in people with celiac disease or NCGS. ATIs resist proteolytic digestion. ATIs are about 2%–4% of the total protein in modern wheat and are present in commercial gluten. A 2017 study in mice demonstrated that ATIs exacerbate preexisting inflammation and may also worsen it at extraintestinal sites.