all aspects of the study were conducted in accordance with the declaration of helsinki and received a favorable ethical opinion from the surrey research ethics committee and the university of surrey ethics committee . they then attended a prescreening session at the university of surrey , typically within 1 month of the laboratory session and during the early afternoon . bmi and waist circumference were measured , and fasting blood samples were provided for measurement of plasma hba1c , insulin and glucose concentrations , and homeostasis model assessment of insulin resistance ( homa - ir ; calculated using homa calculator version 2.2 software [ diabetes trial unit ; university of oxford , oxford , u.k . ] ) . twenty - seven male volunteers were recruited ( 8 lean , 11 overweight , and 8 overweight with type 2 diabetes ) . of the patients with type 2 diabetes , average time since diagnosis was 6.9  2.3 years ; three type 2 diabetic patients were controlled by diet and exercise , and the other five patients were treated with combinations of metformin , statins , ramipril , and lisinopril . one participant within the lean group was a smoker and was required to refrain from smoking for 1 week prior to the study . none of the participants had undertaken shift work within 5 years of the study or crossed any time zones within 1 month of the study . for 1 week prior to the laboratory study , volunteers were required to maintain prescribed daily feeding times and sleep activity ( sleep 22300630 h ) , which were monitored using wrist actigraphy ( awl ; cambridge neurotechnology , cambridge , u.k . ) , sleep diaries , food diaries , and recorded messages on a laboratory time - stamped answer telephone . participants also were required to refrain from eating fatty or sugary foods and drinking alcohol or caffeine throughout the week . for the final 3 days of this baseline week , food was provided by the research team to enable control of timed behavior together with both the quantity and quality of nutrient intake . during these 3 days , the daily caloric content of the supplied food was basal metabolic rate times 1.5 with ~35% of energy from fat . volunteers arrived in the afternoon of day 0 for a night of adaptation . throughout the 2-day study they were required to remain awake with lights on between 0630 and 2230 h ( 440825 lux in direction of gaze ) and allowed to sleep with lights off between 2230 and 0630 h ( 0 , participants were fed with hourly nutritional drinks ( fortisip ; nutricia , schiphol , the netherlands ) and were allowed to drink water ad libitum . daily energy intake was basal metabolic rate times 1.1 , divided equally over the waking hours . four subcutaneous wat biopsies were taken under a local anesthetic ( lidocaine ) from four different sites of the upper buttock region of each participant at 6-hourly intervals for 24 h , beginning at 1030 h on day 1 . wat biopsies were washed with saline and snap - frozen in liquid nitrogen before storage at 80c . cdna was synthesized , and expression was measured for genes integral to the circadian clock ( per1 , per2 , per3 , cry2 , bmal1 , and dbp ) and involved in metabolic activity ( reverb , rip140 , and pgc1 ) by quantitative taqman real - time pcr , as described previously ( 21 ) ( table 1 ) . standard curves ( r > 0.99 ) were generated using human genomic dna ( promega , southampton , u.k . ) , and expression of all genes was normalized to gapdh , as in previous studies of adipose clock gene expression ( 15,16 ) . taqman pcr primer probe sets prescreen data were analyzed using one - way anova with tukey s post hoc test . correlation analyses were conducted using linear regression , and the gene expression time course from biopsies was performed using one - way or two - way repeated - measures anova ( factors time and group ) with tukey s post hoc test . volunteers arrived in the afternoon of day 0 for a night of adaptation . throughout the 2-day study they were required to remain awake with lights on between 0630 and 2230 h ( 440825 lux in direction of gaze ) and allowed to sleep with lights off between 2230 and 0630 h ( 0 lux ) . during the waking period , participants were fed with hourly nutritional drinks ( fortisip ; nutricia , schiphol , the netherlands ) and were allowed to drink water ad libitum . daily energy intake was basal metabolic rate times 1.1 , divided equally over the waking hours . four subcutaneous wat biopsies were taken under a local anesthetic ( lidocaine ) from four different sites of the upper buttock region of each participant at 6-hourly intervals for 24 h , beginning at 1030 h on day 1 . wat biopsies were washed with saline and snap - frozen in liquid nitrogen before storage at 80c . cdna was synthesized , and expression was measured for genes integral to the circadian clock ( per1 , per2 , per3 , cry2 , bmal1 , and dbp ) and involved in metabolic activity ( reverb , rip140 , and pgc1 ) by quantitative taqman real - time pcr , as described previously ( 21 ) ( table 1 ) . standard curves ( r > 0.99 ) were generated using human genomic dna ( promega , southampton , u.k . ) , and expression of all genes was normalized to gapdh , as in previous studies of adipose clock gene expression ( 15,16 ) . prescreen data were analyzed using one - way anova with tukey s post hoc test . correlation analyses were conducted using linear regression , and the gene expression time course from biopsies was performed using one - way or two - way repeated - measures anova ( factors time and group ) with tukey s post hoc test . prescreen data are shown in table 2 . despite efforts to age - match the participants , there was a significant ( p < 0.05 ) difference in age , with post hoc analysis revealing a significant difference only between the overweight and type 2 diabetic groups . however , subsequent analysis using age as a covariant revealed that age , per se , did not alter gene expression ( data not shown ) . there were significant ( p < 0.05 ) differences in bmi , waist circumference , fasting plasma glucose and insulin concentrations , hba1c levels , and homa - ir , with highest values occurring in participants with type 2 diabetes . there was no significant difference in plasma insulin , glucose , hba1c , or homa - ir between the lean and overweight / obese groups , indicating that our overweight / obese participants were insulin - sensitive . participant prelaboratory data * p < 0.05 vs. overweight / obese group . p < 0.05 vs. lean group . in lean individuals , there was a significant ( p < 0.05 ) effect of time on the expression of multiple genes involved in circadian ( per1 , per2 , per3 , cry2 , bmal1 , and dbp ) and metabolic functions ( reverb , rip140 , and pgc1 ) ( fig . minimal expression of reverb and dbp occurred around the middle to end of the afternoon . in keeping with circadian gene expression in other tissues ( 10 ) , the phase of per1 , per2 , per3 , and cry2 was slightly delayed compared with reverb and dbp , with minimal expression occurring around the evening light  dark transition . the bmal1 rhythm was in approximate antiphase to the other circadian genes , with maximal expression around the evening light  dark transition . data are presented as means  se of n = 811 values , normalized to glyceraldehyde 3-phosphate dehydrogenase . there was a significant effect of time ( p < 0.05 , one - way repeated - measures anova ) on the expression of each gene . horizontal bars represent wake ( white ) and sleep ( black ) periods during the laboratory study . zeitgeber time 0 represents the time of lights on ( equivalent to clock time 0630 h ) . analysis by two - way repeated - measures anova of the data from all of the experimental groups confirmed a significant ( p < 0.05 ) effect of time on gene expression ( fig . however , it is surprising that we did not observe a significant effect of experimental group on the expression of any gene . the only gene to exhibit a significant ( p < 0.01 ) time  group interaction was bmal1 . post hoc analysis revealed that bmal1 expression 16.5 h after lights on ( equivalent to clock time 2300 h ) was significantly ( p < 0.05 ) lower in the overweight / obese participants than in the lean or type 2 diabetic groups . comparison of 24-h gene expression profiles in human wat from individuals who are lean ( solid line ,  ) , overweight / obese ( dashed line ,  ) , or overweight / obese with type 2 diabetes ( dotted line ,  ) . data are presented as means  se of n = 811 values , normalized to glyceraldehyde 3-phosphate dehydrogenase . for all genes , there was a significant ( p < 0.05 ) effect of time but no subject group effect on gene expression ( two - way repeated - measures anova ) . with the exception of bmal1 ( p < 0.01 ) horizontal bars represent wake ( white ) and sleep ( black ) periods during the laboratory study . zeitgeber time 0 represents the time of lights on ( equivalent to clock time 0630 h ) . consistent with a previous study ( 22 ) , our data reveal robust diurnal rhythmicity within human wat in vivo . to date , limited data have been published concerning clock gene expression in this tissue . because of difficulties in acquiring human tissue , some studies have measured clock gene expression in wat collected at a single time point ( 18,19 ) , although it is clearly difficult to infer rhythmical changes from such a temporally limited dataset . an elegant compromise has been to culture explants of tissue taken at a single time point and then measure gene expression in fragments of the biopsies over a 24-h time course ( 17 ) . although such studies highlight endogenous wat rhythms , they do not reflect gene expression in vivo . therefore , we measured gene expression in human tissue explants harvested across a whole 24-h cycle to provide in vivo human wat rhythms . animal models suggest that obesity and type 2 diabetes reduce rhythm amplitude in murine wat because lean c57bl/6j mice exhibit higher amplitude wat rhythms than obese / diabetic kk and kk - a mice ( 15 ) . other data revealed reduced amplitude rhythms in mice that became obese as a result of a high - fat diet ( 16 ) . however , ando et al . ( 15 ) compared mice with differing genetic backgrounds , which complicate interpretation of their data , and it is possible that dietary intervention could directly regulate gene expression rather than obesity per se . in contrast to the above studies , we observed minimal differences between clock gene rhythms in human wat . although murine bmal1 has been implicated in the control of adipogenesis in vitro ( 23 ) , juvenile bmal1 mice develop adipose depots comparably to their wild - type littermates ( 24 ) . furthermore , the similarity between gene expression profiles in lean individuals and those who were overweight with type 2 diabetes suggests that bmi is not the cause of the time  group interaction for bmal1 in the overweight , nondiabetic individuals . the similarity of gene expression profiles between our experimental groups may partly result from a lack of extreme phenotypic differences . although there was not a large disparity in bmi between our groups , all participants fell into the current clinical guidelines for lean / healthy , obese , and type 2 diabetic individuals . therefore , there was no effect of obesity or type 2 diabetes , per se , on human wat rhythmicity . it remains possible that severely obese individuals may exhibit reduced amplitude wat rhythmicity ; however , interpretation of data from such individuals is complicated by various confounding factors , such as comorbidities associated with type 2 diabetes . another difference between our study and previous work ( 25 ) is the high level of glycemic control in our participants . we specifically aimed to investigate the relationship between wat rhythmicity , body weight , and presence of type 2 diabetes . diurnal adipose gene expression may be modulated by the level of glycemic control and/or drugs taken by diabetic individuals . alternatively , the association between metabolic state and wat rhythmicity might vary between adipose depots . however , because of limitations of sampling and experimental group size , we could not directly address these possibilities in the current study . the persistence of 24-h rhythms in wat from patients with type 2 diabetes now suggests that the link between human circadian and metabolic physiology occurs outside of wat , at least in the earlier stages of the disease .