We recruited 10 healthy participants aged 20–55 years (six male, four female) for four repeated multiple-sampled OGTTs with insulin assays. ‘Healthy’ was defined as no acute or chronic injury or illness requiring medical attention in the previous 3 months and a current HbA_1c < 40 mmol/mol (5.8%). These tests were conducted according to the protocols outlined by Kraft⁹ and standard oral glucose tolerance testing procedures, including an overnight fast and no vigorous exercise on the morning of the test. Participants were also required to maintain the consumption of at least 150 g carbohydrate per day for at least 14 days prior to the first test and throughout the testing period.⁹ Participants maintained their normal habits, especially physical activity patterns, throughout the 2-week lead-in and 4-week study period. A deliberate decision was made to not formally assess diet or physical activity to make the results more representative of those that would be seen in clinical practice. The overall design including the clinical criteria and short study time meant it would be unlikely that an underlying clinical condition would influence insulin responses and confound the results.

On each test occasion, after an overnight fast, each subject had a cannula inserted into their antecubital fossa and provided fasting venous blood samples before consuming 100 g glucose (400 mL Carbotest™ solution). The glucose was consumed within 10 min of test commencement (0 min). With the exception of water, no further food or drink was permitted until the end of the test. Further venous samples were drawn at 30, 60, 120 and 180 min. Vein patency was maintained by flushing with saline before and after each collection, with the first 2 mL of blood collected being discarded. Blood samples were collected into plasma separator tubes (PST) vacutainers (Becton, Dickinson and Company, Franklin Lakes, NJ) for glucose and insulin analysis. Plasma was extracted from the PST tubes after centrifugation (1500 × g at 4°C for 10 min), and then frozen at -20°C within 2 h of collection. This protocol was repeated weekly for a total of four tests. On the first test occasion, height, weight and waist girth (smallest girth between iliac crest and the lowest rib) were measured. On the initial testing occasion, an additional fasting venous blood sample was collected into an Ethylenediaminetetraacetic acid (EDTA) vacutainer (Becton, Dickinson and Company) for HbA_1c analysis.

This study was granted ethical approval by Auckland University of Technology Ethics Committee (AUTEC) on 16 December 2014 (reference no. 14/363).

Mean–variance relationships could only be detected for fasting glucose, fasting insulin and glucose at 180 min. After the removal of participant K4 from the data set, a mean–variance relationship could no longer be detected for either fasting glucose or glucose at 180 min. Log-transformation of fasting insulin did not remove the mean–variance relationship. No mean–variance relationship could be detected for fasting insulin for the subset of hyperinsulinaemic participants (K2, K5 and K6).

Table 2³ displays the RepCoef for all time points for insulin and glucose, the McAuley Index, all HOMA measures and OGIS. With the exception of glucose 0 min, there was no practical difference in the RepCoef for glucose when participant K4 was excluded. Among the fasting models of insulin resistance, the McAuley Index had the lowest RepCoef compared to the grand mean of the sample (17.4%).

Table 3³ presents the distribution of each test per participant for both Kraft and Hayashi insulin response patterns. The most common Kraft pattern was pattern I, recorded by five of the eight participants on at least one occasion, while the most common Hayashi pattern was pattern 3, which was recorded by every participant on at least one occasion. No participant recorded a Kraft IV or V pattern, or a Hayashi pattern 5.

Three participants (K5, K6 and K10) were initially excluded from κ calculations as they did not have four eligible tests for both pattern responses. However, small participant numbers meant that missing data decreased the power of the study. Therefore, we replicated the repeatability calculations after imputing the clinically most likely, or most frequent, outcome for participants with missing data (K5, K6 and K10), as shown in Table 4³ and Table 5³.

The inclusion of the imputed data did not cause a qualitative change in the overall results, as shown in Table 6³. Estimated κ for the Kraft patterns was higher than for Hayashi patterns (0.290 vs. 0.186), but only the κ for the Kraft patterns was significantly different from zero (95% CIs, 0.515–0.798 and −1.238 to 1.610 for Kraft and Hayashi, respectively).

Table 7³ displays the participants’ insulin resistance measures when dichotomised into normal (Kraft I) and hyperinsulinaemic (Kraft IIA, IIB, III) insulin response patterns.⁹ Following a two-sample t test (defined by Kraft pattern), statistically significant differences can be noted for HOMA2 measures and for OGIS, but not for the McAuley Index.

Of the insulin resistance measures (HOMA2, McAuley and OGIS), only the McAuley Index (fasting measure) and OGIS (dynamic measure) demonstrated a low RepCoef relative to the grand mean of the sample population with a change of 17.4% and 14.8%, respectively. By contrast HOMA2 variants had a greater degree of change (HOMA2 %B = 41.3%, HOMA2 %S = 55.9% and HOMA2 IR = 75.4%). These HOMA2 findings are comparable to our previous research in a population of people with normal glucose tolerance.¹¹

Most studies assess repeatability using CV. Although it may not be possible to directly compare the repeatability of the original HOMA model with the HOMA2 model, our findings (HOMA2 %B = 14.8%, HOMA2 %S = 20.1% and HOMA2 IR = 27.1%) align with CVs reported from the original model including that of Mather and colleagues, who reported HOMA IR having a CV of 24%.²⁰ Coefficient of variation data for the McAuley Index is limited with one study reporting a CV of 15.1%.¹³ This is higher than our finding of 6.3%.

There is limited data on the repeatability of the OGTT, yet it is a very common clinical test.²¹ Few studies have investigated the repeatability or reproducibility of insulin response curves; of those that have, no significant differences in AUC_insulin have been noted.^21,22 There are no published studies that have assessed the repeatability of insulin response patterns, namely the Kraft and Hayashi patterns. Our study demonstrated that the Kraft pattern methodology had a higher reproducibility and was more likely to be consistent following repeated OGTT when compared to the Hayashi patterning method. Kraft patterns account for both the magnitude of the insulin response and rate of decay as well as the timing of the insulin peaks. By contrast, the Hayashi pattern algorithm is based solely on the timing of the insulin peaks. As there is little long-term data associating insulin response patterns to health outcomes, we suggest that insulin response patterns should be categorised using a combination of factors, including the magnitude and timing of the insulin peak and rate of decay.

Consistency among insulin response patterns was more common for participants who were predominantly Kraft I pattern (n = 5). Two participants deviated from Kraft I pattern for one of the four tested occasions: K4 (week 1) and K10 (week 4). It is unknown why these deviations occurred. Changes to insulin responses can occur for a variety of reasons, including sub-acute illnesses, menstrual cycle, stress or even poor sleep patterns.^8,23,24 This suggests that insulin response patterning should only be conducted during times of stable clinical condition or with an understanding of these caveats. Concurrent assessment of inflammatory markers such as c-reactive protein or cortisol could also be considered.

For those participants who never exhibited a Kraft I pattern (n = 3), consistency among patterns was lower. Two participants exhibited a 50:50 split between patterns IIB and III, while the third was predominately pattern IIB, with one occasion of pattern IIA. Unlike the participants who deviated from a predominant Kraft I pattern, there was no clear plausible clinical indication for these variations. These fluctuations may indicate that hyperinsulinaemic states are more transitory than a normal insulin response (Kraft I) and they support previous findings that demonstrate that people with hyperinsulinaemia have lower repeatability rates with OGTT.²¹ Assessing insulin pattern repeatability in a large cohort of people with known hyperinsulinaemia is required to understand these variations. This study suggests that Kraft patterns are sufficiently reproducible to dichotomise patients into a Kraft I pattern or ‘normal’ insulin status, or a hyperinsulinaemic status (Kraft patterns II–III); however, a larger study is required to confirm these results.

Variation was higher within the Hayashi patterns. Every participant exhibited a Hayashi 3 pattern at least once. Most (75%) also exhibited either a Hayashi 1 or 2 pattern, or a Hayashi 4 pattern. With one exception, no participant had both a Hayashi 1 or 2 pattern and a Hayashi 4 pattern. Although this increased variation within the Hayashi patterns suggests that Kraft patterns should be preferred to Hayashi patterns in future research, it must also be noted that Kraft patterns, to date, do not have any longitudinal outcome data.

Using the definition of normal insulin tolerance as Kraft I pattern,⁹ the McAuley Index was unable to distinguish between normal and hyperinsulinaemic sub-groups. This contrasts to the HOMA2 variables and OGIS, which all had clear delineations between the normal and hyperinsulinaemic sub-groups. Returning a similar value across a range of Kraft patterns, HOMA2 and OGIS values suggests the McAuley Index is less sensitive to changes of physical state than the other measures.

Although HOMA2 variants clearly delineated between normal and hyperinsulinaemic states, high variability decreases the sensitivity of the test. Only OGIS had both sensitivity and repeatability. This further questions the value of fasting tests, especially for assessing compensatory hyperinsulinaemia. Our previous research found a poor association between a fasting insulin < 30 µU/mL and a delayed insulin peak.¹¹

We recognise that our study had a number of limitations, especially with respect to participant dropout rates and small sample size. However, sample sizes of 10 participants are common in repeatability studies for insulin resistance.^21,25 Nevertheless, this study may be the first to assess the test–retest repeatability of insulin response patterns. Future research for diagnosing insulin resistance should focus on a dynamic test based on an OGTT. There are concerns about using methodologies based on the oral glucose tests because of previous reports of poor repeatability or variable glucose absorption rates. However, our study has shown that dynamic tests have a higher degree of repeatability compared to those based on fasting models. The lower rate of repeatability from models based on fasting tests may be because of the natural lability of insulin, which, our study shows, has a CV of 25% – a figure consistent with previous reports.²⁶

Although previous research has focused on diagnosing insulin resistance for the early diagnosis of many metabolic diseases, hyperinsulinaemia is an emerging field.^3,27 Although there is an accepted association between insulin resistance and hyperinsulinaemia, the direction of causality is unknown and there are multiple plausible aetiologies that could start with either condition.^3,27 It is also becoming accepted that hyperinsulinaemia may be corrected while insulin resistance is maintained. Given the high degree of overlap between the conditions, it is also plausible that diagnostic tests for hyperinsulinaemia and insulin resistance may overlap. Given the variability of fasting insulin, this study suggests that dynamic insulin or glucose modelling or insulin response patterning may be more effective in diagnosing hyperinsulinaemia and/or insulin resistance, and this is where future research should be focused.