the eurodiab study recruited 3,250 patients with type 1 diabetes ( 1,668 men and 1,582 women ; mean  sd age 32.7  10.2 years and duration of diabetes 14.7  9.3 years ) . these participants were selected at random from age , diabetes duration , and sex strata from 31 diabetes clinics across europe . twenty - seven centers participated in the follow up of the eurodiab cohort ( the eurodiab prospective complications study [ eurodiab pcs ] ) . we present data collected in the 13 centers that participated in the nerve conduction substudy , conducted during the follow - up examinations ( 19971999 ) . patients were randomly selected by the eurodiab data coordinating center , and a list of selected patients was sent to the local main investigator . selected patients were called for the nerve conduction study after they completed the main eurodiab pcs follow - up examination . we excluded 44 patients because of incomplete data on cardiovascular risk factors ; 130 patients were excluded because of incomplete nerve conduction data , 91 of these were only missing data from the sural nerve . data from these 91 patients were initially included in analyses of the peroneal and ulnar nerves , but these results did not differ markedly from a complete data analysis ( data not shown ) . six patients were excluded because of extreme outlying values on nerve conduction tests , yielding a set of 456 patients with complete data . it is likely that some of these data were missing because the true value of nerve conduction was below the threshold of detection ( i.e. , the true nerve conduction value is not zero / missing , but rather too low to be measured ) ; thus , we supplemented the complete - case analyses , on which this article is based , with analyses using multiple imputation techniques and single imputation ( using the first percentile of nerve conduction values as imputed value ) to explore the impact of missing values on our results . we report primary results based on complete - case analysis ( 456 patients ) and provide results from the supplementary multiple and single imputation analysis for reference ( supplementary methods , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc10-0456/dc1 ) . these were sent to a central laboratory and analyzed for a1c , total and hdl cholesterol , and triglycerides ( 11 ) . ldl cholesterol was calculated . to compare the results with those of the diabetes control and complications trial ( dcct ) , measured a1c values were converted as reported previously ( 11 ) and used in all subsequent analyses . a urinary albumin excretion rate of 20200 g / min was defined as microalbuminuria ; a rate > 200 g / min was defined as macroalbuminuria . the presence and severity of diabetic retinopathy were assessed from retinal photographs ( two fields per eye ) obtained with a wide - angle camera and scored centrally . cardiovascular disease ( cvd ) was defined as either a history of physician - diagnosed cvd ( e.g. , previous myocardial infarction , angina , coronary - artery bypass grafting , or stroke ) or ischemic changes detected on a 12-lead electrocardiogram ( classified by two observers according to the minnesota code ) . clinical neuropathy was diagnosed in patients with two or more of the following four measures : the presence of one or more symptoms , the absence of two or more reflexes of the ankle or knee tendons , a vibration perception threshold that was abnormal for the patient 's age , and abnormal autonomic function ( loss of heart rate variability with a ratio of < 1.04 , postural hypotension with a fall in systolic blood pressure of 20 mmhg , or both ) . the cutoff points and the definition of neuropathy were established before any examination of the outcome data . the ncss were performed with standard electrophysiological and electromyographic equipment provided by major manufacturers ( e.g. , nicolet , teca , and disa ) . digital skin thermometers accurate to 0.1c and , when necessary , appropriate warming measures were used to ensure that skin temperatures were at least 32c . examinations were performed by trained electrophysiologists with extensive experience in recording and interpreting clinical data . velocity was determined for the distal sural and ulnar sensory nerves and for the peroneal motor nerve ( knee to ankle ) unilaterally on the nondominant side . response amplitude was measured overlying the dorsal surface of the foot for the sural nerve , at the fifth finger for the ulnar nerve and overlying the extensor digitorum brevis muscle for the peroneal nerve . all data were collected using standard surface techniques and specified anatomical landmarks for both stimulation and recording sites . before testing of study participants , all participating electrophysiologists attended a standardization training meeting , and all centers were certified by submitting three satisfactory complete examinations using specific study procedures by the central reading site ( j.c.a . at the albert einstein college of medicine , new york , ny ) . throughout the study , a sample of approximately one - third of the data from each center was reviewed by the central reading site to ensure overall quality and continued adherence to study procedures . statistical analyses were performed with stata software ( version 11 ; statacorp , college station , tx ) . characteristics of patients who did and did not participate in the ncss were compared with the use of the student t test or mann - whitney u test where appropriate . in the case of unequal variances , means were compared by approximate inference based on the t distribution . multivariate linear regression models were used to describe the associations among cardiovascular risk factors , anthropometric measures , the presence / severity of complications , and ulnar , sural , and peroneal nerve conduction , which are reported as unstandardized  coefficients . data were collected as crude data from each center ; therefore , models for nerve conduction measures were adjusted for age , sex , height , diabetes duration , a1c , center , skin temperature , and distance between electrodes into the model . these covariates were prespecified as factors that may affect nerve conduction based on previously published evidence ( 13,14 ) . nerve conduction velocity ( ncv ) and nerve conduction amplitude ( nca ) for the three nerves were standardized and summarized per individual in a total z score , which was used as an additional outcome in a separate regression model . a slow / low nerve conduction measure was defined as being in the lowest tertile of ulnar , sural , and peroneal ncv or nca , and odds ratios ( ors ) for being in this group were calculated using a multivariate logistic regression model with adjustment for the sample variables as mentioned above . blood samples were obtained ideally after an overnight fast . these were sent to a central laboratory and analyzed for a1c , total and hdl cholesterol , and triglycerides ( 11 ) . ldl cholesterol was calculated . to compare the results with those of the diabetes control and complications trial ( dcct ) , measured a1c values the urinary albumin excretion rate was the average from two 24-h urine collections . a urinary albumin excretion rate of 20200 g / min was defined as microalbuminuria ; a rate > 200 g / min was defined as macroalbuminuria . the presence and severity of diabetic retinopathy were assessed from retinal photographs ( two fields per eye ) obtained with a wide - angle camera and scored centrally . cardiovascular disease ( cvd ) was defined as either a history of physician - diagnosed cvd ( e.g. , previous myocardial infarction , angina , coronary - artery bypass grafting , or stroke ) or ischemic changes detected on a 12-lead electrocardiogram ( classified by two observers according to the minnesota code ) . clinical neuropathy was diagnosed in patients with two or more of the following four measures : the presence of one or more symptoms , the absence of two or more reflexes of the ankle or knee tendons , a vibration perception threshold that was abnormal for the patient 's age , and abnormal autonomic function ( loss of heart rate variability with a ratio of < 1.04 , postural hypotension with a fall in systolic blood pressure of 20 mmhg , or both ) . descriptions of the specific tests have been published previously ( 12 ) . the cutoff points and the definition of neuropathy were established before any examination of the outcome data . the ncss were performed with standard electrophysiological and electromyographic equipment provided by major manufacturers ( e.g. , nicolet , teca , and disa ) . digital skin thermometers accurate to 0.1c and , when necessary , appropriate warming measures were used to ensure that skin temperatures were at least 32c . examinations were performed by trained electrophysiologists with extensive experience in recording and interpreting clinical data . velocity was determined for the distal sural and ulnar sensory nerves and for the peroneal motor nerve ( knee to ankle ) unilaterally on the nondominant side . response amplitude was measured overlying the dorsal surface of the foot for the sural nerve , at the fifth finger for the ulnar nerve and overlying the extensor digitorum brevis muscle for the peroneal nerve . all data were collected using standard surface techniques and specified anatomical landmarks for both stimulation and recording sites . before testing of study participants , all participating electrophysiologists attended a standardization training meeting , and all centers were certified by submitting three satisfactory complete examinations using specific study procedures by the central reading site ( j.c.a . at the albert einstein college of medicine , new york , ny ) . throughout the study , a sample of approximately one - third of the data from each center was reviewed by the central reading site to ensure overall quality and continued adherence to study procedures . statistical analyses were performed with stata software ( version 11 ; statacorp , college station , tx ) . characteristics of patients who did and did not participate in the ncss were compared with the use of the student t test or mann - whitney u test where appropriate . in the case of unequal variances , means were compared by approximate inference based on the t distribution . multivariate linear regression models were used to describe the associations among cardiovascular risk factors , anthropometric measures , the presence / severity of complications , and ulnar , sural , and peroneal nerve conduction , which are reported as unstandardized  coefficients . data were collected as crude data from each center ; therefore , models for nerve conduction measures were adjusted for age , sex , height , diabetes duration , a1c , center , skin temperature , and distance between electrodes into the model . these covariates were prespecified as factors that may affect nerve conduction based on previously published evidence ( 13,14 ) . nerve conduction velocity ( ncv ) and nerve conduction amplitude ( nca ) for the three nerves were standardized and summarized per individual in a total z score , which was used as an additional outcome in a separate regression model . a slow / low nerve conduction measure was defined as being in the lowest tertile of ulnar , sural , and peroneal ncv or nca , and odds ratios ( ors ) for being in this group were calculated using a multivariate logistic regression model with adjustment for the sample variables as mentioned above . characteristics of the patients included in the current analyses compared with those of all the eurodiab participants who completed the epcs follow - up are shown in table 1 . patients in the ncs analysis set were younger ( 36.8 years ) , had a shorter diabetes duration ( 19.5 years ) , and had more favorable levels of most cardiovascular risks factors and a lower prevalence of complications . we compared the patients excluded from our ncss because of missing values and found that they were older ( 43.9 years ) , had longer diabetes duration ( 25.2 years ) , and had higher levels of all cardiovascular risk factors and complications . unadjusted mean ncv and nca values for participants by complication status are presented in table 2 . the associations among cardiovascular risk factors , anthropometric measures , diabetes complications , and ncv / nca are shown in supplementary tables 1a and 2a ( available in an online appendix ) . comparison with results from the imputation analyses of velocity / amplitude z scores are shown in supplementary tables 3a and 4a ( available in an online appendix ) . coefficients denote the difference in ncv / nca per unit difference in each determinant ; e.g. , for a 1-year increment in age , ulnar ncv is 0.15 m / s lower . characteristics of all participants of the eurodiab pcs and those who were included in the nerve conduction substudy analysis data are means  sd , skewed distribution : median ( 5th ; 95th percentile ) , or n ( % ) unless otherwise indicated . ncss of ulnar and distal sural sensory nerves and the peroneal motor nerve ( knee to ankle ) unilaterally on the nondominant side in the eurodiab pcs at follow - up data are means  sd and are unadjusted from the complete - case analysis . we confirm that duration of diabetes and a1c were both negatively and statistically significantly associated with ncv and nca in all three nerves . we also confirm that the presence of nephropathy or retinopathy or a clinical diagnosis of neuropathy was associated with low ncv and nca z scores . weight was associated with a higher ncv z score , but this effect was not observed for nca . the association between height and lower ncv or nca was only present in the nerves of the lower extremity . ors for having a z score in the lowest tertile of ncv / nca by diabetes complication status are shown in fig . microalbuminuria was associated with increased odds of low ncv or nca ( or 2.25 [ 95% ci 1.154.40 ] and 2.29 [ 1.124.66 ] , respectively ) . in the case of nonproliferative retinopathy , values were 2.30 [ 1.134.67 ] and 2.29 [ 1.154.58 ] for ncv and nca , respectively . a similar increase in the odds of low ncv or nca was found for each 2% difference in a1c ( 2.39 [ 1.683.41 ] and 2.47 [ 1.723.54 ] , respectively ) or a difference of 10 years of duration of diabetes ( 3.09 [ 2.094.57 ] and 3.01 [ 2.034.47 ] , respectively ) . we repeated our analyses on a subset of the patients without clinical signs of neuropathy . this restriction did not materially affect our findings ( data not shown ) . or of the lowest tertile of the velocity ( left ) and amplitude z score ( right ) , adjusted for age , sex , height , center , diabetes duration , a1c , and determinants of nerve conduction measures ( skin temperature and distance between electrodes ) . z scores are calculated from a summed score of standardized ncv / nca from ulnar and distal sural sensory nerves and the peroneal motor nerve ( knee to ankle ) unilaterally on the nondominant side . * microalbuminuria ( microalb ) : 20200 g / min ; macroalbuminuria ( macroalb ) > 200 g / min . * * prevalence of cvd . composite score of symptoms , absent reflexes , abnormal vibration perception threshold , or autonomic dysfunction . results from the subsidiary analyses using imputed datasets yielded results that were broadly similar to those for the complete - case analysis ( supplementary tables 1a and 2a ) . we showed in one of the largest sets of patients with type 1 diabetes to date that , in addition to duration of diabetes and level of a1c , the presence of other microvascular diabetes complications is associated with low ncv and nca . we did not find associations with blood pressure , lipid levels , bmi , waist - to - hip ratio , smoking , or alcohol consumption . the eurodiab pcs offers the possibility to study complications of type 1 diabetes , because it provides a large , multicenter , european cohort of patients attending a clinic at least once a year . therefore , compared with other studies , our study on nerve conduction has the strength of containing extensive information on these measures for 634 patients with type 1 diabetes , and we have the ability to take this information into account in our analyses . complete - case analysis could therefore be subject to a degree of selection bias because the term  missing could reasonably be applied to one of three circumstances : 1 ) the specific nerve was never tested ( e.g. , the patient did not permit testing of the ulnar nerve due to pain ) ; 2 ) data were collected , but judged to be technically unacceptable ( e.g. , the sensory response was distorted by a prolonged stimulus artifact ) ; or 3 ) correct procedures were followed , but the nerve response was below the detection thresholds using the applied surface recording procedures ( i.e. , patients with advanced neuropathy have an increased risk of obtaining a missing value ) . to explore the effect of missing values , we performed subsidiary analyses using single or multiple imputation methods . multiple imputation is best applied when data are missing at random , a condition that is demonstrably not met in our data . single imputation analysis , which assigns the lowest observed ncs values to those with a missing value , does not deal with the variance structure of the imputed values and may therefore lead to incorrectly narrow cis . because neither imputation method is satisfactory in our dataset , we focused on the complete - case analysis and provide the results of the subsidiary imputation analyses as reference . it is important to note that our main results and conclusions were not dependent on the chosen statistical method , indicating that the observed associations are unlikely to have been introduced by participant selection . it is known that age , sex , and height have major effects on nerve conduction in healthy individuals ( 13,14 ) . these associations also exist in our population of type 1 diabetic patients , but we can not investigate from our data whether the association goes beyond the effects in health . our study is based on a population of european caucasian patients with type 1 diabetes , and one can not readily generalize these findings to a broader diabetes population . it is possible that different associations or effect magnitudes are applicable among individuals with type 2 diabetes or in other populations . the patients included in our analysis were younger and had fewer potential risk factors than the rest of the eurodiab population , but because the findings of imputation analysis confirm the findings from the complete - case analysis , we are confident that our conclusions are representative of the eurodiab population and that the mechanisms of neuropathy development are likely to be similar for a majority of patients . a direct comparison of different covariates on nerve conduction will increase the clinician 's ability to identify those patients most likely to have neuropathic damage . other studies have previously shown that duration of diabetes and glycemic control are related to nerve conduction measures . the dcct showed the importance of glycemic control in the development of neuropathy and also showed that even in patients without diagnosable neuropathy there was a relation between glycemic control and nerve conduction ( 15 ) . a cross - sectional analysis of 162 patients with type 1 diabetes and 267 patients with type 2 diabetes in the early diabetes intervention trial found that age , sex , and anthropometric factors were related to nerve conduction measures , with no difference by diabetes type ( 16 ) . a smaller study of 25 patients with type 1 diabetes and 72 with type 2 diabetes found an association among glycemic control , male sex , diabetes duration , and height and lower ncv or nca , but no impact of weight , systolic and diastolic blood pressure , neuropathy duration , and plasma cholesterol or triglyceride levels ( 17 ) . our study confirms the association with male sex , glycemic control , and diabetes duration on objective measures of nerve conduction . several of the smaller studies performed to date have examined the effect of cardiovascular risk factors on nerve conduction . some found no impact of weight , systolic and diastolic blood pressure , neuropathy duration , and plasma cholesterol or triglyceride levels ( 17 ) but also suggested that vascular factors participate in the development of neuropathy ( 18 ) , whereas others reported an independent association between pulse pressure and hypertension and diabetic peripheral neuropathy in patients with type 2 diabetes ( 19 ) . a prospective study of 57 patients with type 2 diabetes did not confirm these findings ( 20 ) . an association between elevated triglycerides and sural nerve myelinated fiber density has been reported ( 21 ) . because of their small size , these studies had a limited ability to adjust for potential confounding . based on our findings , there does not seem to be a clear association of ncss with cardiovascular risk factors , taking important covariates into account , and therefore the level of cardiovascular risk factors can not help a clinician in selecting patients with neuropathic damages . the strong association among retinopathy , nephropathy , and neuropathy has been noted by many others ( 1 , 4 , 9 , 22 , 23 ) . a substudy from the rochester diabetic neuropathy study ( rdns ) cohort , involving 238 patients with diabetes ( 80% type 2 diabetes ) , found that subclinical nerve dysfunction precedes the diagnosis of polyneuropathy and showed that a composite score of nerve conduction abnormality was unequivocally superior to clinical impairment , symptoms , or quantitative sensation testing in identifying patients with monotone worsening ( 6 ) . the rdns further showed that 24-h microalbuminuria was a significant risk factor for worsening of nerve conduction among patients with diabetes ( 6 ) and that microvessel disease , chronic hyperglycemic exposure , and type of diabetes were associated with the severity of diabetic polyneuropathy ( 24 ) . our findings confirm that there is a close relation between the presence of microvascular complications and reduced ncv / nca , indicating that the deleterious microvascular effects of long - term exposure to hyperglycemia develop in parallel , even if the clinical presentation of symptoms may suggest a sequence . in the eurodiab population however , there was only a weaker association between the development of neuropathy and other markers of microvessel disease ( 9 ) . we considered several potential explanations for our finding of no association between cardiovascular disease and ncs results . although the patients in our analysis may not be fully representative of the complete eurodiab population , this selection is unlikely to affect the direction and magnitude of risk factor associations within the substudy . we performed a subanalysis excluding those with a positive neuropathy score at baseline and still found no association with cvd . it is conceivable that our assessment of cvd , which is based on physician - diagnosed cvd and verified ischemic changes on the electrocardiogram , lacks the precision needed to detect subtle associations with ncss . however , we believe that our method may have a low specificity but has a good sensitivity to detect cvd . we found that 35 of 456 patients had cvd according to our definition , whereas 11 had macroalbuminuria and 47 had proliferative retinopathy . any true association that we may have missed is therefore likely to be of small magnitude compared with those seen with the microvascular complications . this finding supports the theory that there is a common pathophysiological pathway of microvessel disease , which has no direct relation to macrovascular disease . because we did not find clear associations with other cardiovascular risk factors , the pathogenic effects of hyperglycemia appear to be the main etiological factor . however , this is a cross - sectional study , and thus we can not infer any causal connection to risk factors . a prospective study with detailed assessment of microvascular pathophysiology and ncs progression from baseline would be necessary to answer this question . it has been documented that neuropathy often is subclinical ( 15,25 ) . based on our finding of close associations among markers of microvessel disease , the finding of any indication of incipient microvessel disease should therefore alert the clinician toward thinking that the patient has an increased risk of having neuropathy . therefore , if such a patient does not show signs of neuropathy on the clinical neurological assessment , referral for a ncs may be worth considering .
objectiveslow nerve conduction velocity and reduction in response amplitude are objective hallmarks of diabetic sensorimotor polyneuropathy . because subjective or clinical indicators of neuropathy do not always match well with the presence of abnormal nerve physiology tests , we evaluated associations to nerve conduction in patients with type 1 diabetes.research design and methodsnerve conduction studies were performed in the distal sural and ulnar sensory nerves and the peroneal motor nerve in 456 individuals with type 1 diabetes who participated in the follow - up visit of the eurodiab prospective complications study ( epcs ) . 
 we used multivariate regression models to describe associations to decreased nerve conduction measures.resultsin addition to an effect of duration of diabetes and a1c , which were both associated with low nerve conduction velocity and response amplitude , we found that the presence of nephropathy , retinopathy , or a clinical diagnosis of neuropathy was associated with low nerve conduction velocity and amplitude . in the case of nonproliferative retinopathy , 
 the odds ratio ( or ) for being in lowest tertile was 2.30 ( 95% ci 1.134.67 ) for nerve conduction velocity . a similar or 
 was found for each 2% difference in a1c ( 2.39 [ 1.683.41]).conclusionswe show that the presence of other microvascular diabetes complications , together with diabetes duration and a1c , are associated with low nerve conduction velocity and amplitude response and that cardiovascular disease or risk factors do not seem to be associated with these measures .