The potential of biomarkers for diagnosing insomnia: Consensus statement of the WFSBP Task Force on Sleep Disorders

Figures & data

Table 1. Grading system for establishing diagnosis.

Grade of diagnostic pertinence
Grade	Data reported or extracted
A (high)	Sensitivity and Specificity or AUC of the Receiver Operating Characteristic (ROC) curve
B (moderate)	Cohen’s d or Hedge’s g (provided or extracted from available data as shown in Supplementary Table S1)
C (low)	Correlation coefficient (r)* or data from review studies relevant for the assessment of diagnostic validity
D (inadequate)	Data inadequate for the establishment of diagnostic accuracy (p-values, mean values without available standard deviations, geometric mean values)
Grade of diagnostic accuracy
Grade	Sensitivity and specificity	AUC of ROC	Cohen’s d or Hedge’s g	Correlation coefficient (r)
1 (high)	≥0.80	≥0.80	≥1.50	≥0.80
2 (moderate)	0.70–0.79	0.70–0.79	1.00–1.49	0.60–0.79
3 (low)	0.60–0.69	0.50–0.69	0.70–0.99	0.40–0.59
4 (virtually absent)	<0.60	<0.50	<0.70	<0.40

*Between the diagnostic method under examination and another well-established method for diagnosing insomnia.

Table 2. Studies of psychometric measurements in diagnosing insomnia.

Citation	Type of study	Type and number of subjects	Instruments and methods	Results	Main conclusions	Comments
(a) Rating scales and questionnaires for the assessment of insomnia
Buysse et al. (1989)	Case-control; psychometric instrument development (Pittsburg Sleep Quality Index—PSQI)	45 INS; 17 OSD; 34 Psy; 52 HS	PSQI; CΒDI; PSG	Sens 84.4% (for INS)*, 89.6% (for all study pts); Spec 86.5% (all study pts); α = 0.83; T-R = 0.85; Ext val: r = 0.20 (only for SOL) to PSG	The PSQI covers a multitude of sleep complaints/disorders with high scores of reliability and validity. Regarding external validity, correlation to PSG was very low or non-existent. Diagnostic Grading: A1	The PSQI is quite lengthy and its rating procedure is rather complicated; yet, it is widely used in research projects, evidently due to its comprehensiveness and its satisfactory psychometric properties.
Soldatos et al. (2000)	Case-control; psychometric instrument development (Athens Insomnia Scale—AIS)	105 INS; 144 Psy; 50 HS	AIS; CΒDI; SPS	Sens 93%; Spec 85%; PPV 86%; NPV 92%; α = 0.90; T-R = 0.89 (1 week); Ext val: r = 0.90 to SPS, r = 0.76 to PSQI**	The AIS, based on ICD-10 criteria for insomnia, presents with high scores of reliability and validity. Diagnostic Grading: A1	The AIS consists of 8 items and is easy to use and to rate. While not providing information on other sleep disorders apart from insomnia, it is being widely used by clinicians and researchers presumably due to its simplicity and its diagnostic usefulness.
Roth et al. (2002)	Case-control; psychometric instrument development (Global Sleep Assessment Questionnaire—GSAQ)	104 INS; 95 OSD; 13 HS	GSAQ; CΒDI; SF-36; MOS-SPI	Sens 79%; Spec 57%; ROC-AUC = 0.72; T-R = 0.86 (2 weeks)	The GSAQ is a screening tool, especially useful in sleep centres and primary care settings for distinguishing among primary insomnia, insomnia associated with a mental disorder, OSA, PLM and parasomnias. Diagnostic Grading: A2	Sensitivity, specificity and AUC of the GSAQ were relatively low for primary insomnia, while they were generally higher for sleep disorders other than insomnia. A limitation of the study is the small sample size of HS.
Spoormaker et al. (2005)	Case-control; psychometric instrument validation (Sleep—50 Questionnaire)	65 INS; 213 OSD; 380 HS	Sleep-50 Questionnaire; CΒDI; sleep log	Sens 71%; Spec 75%; α = 0.85; T-R = 0.78 (3 weeks)	Sensitivity and specificity of the SLEEP-50 Questionnaire for diagnosing insomnia, although quite satisfactory, are generally lower than relevant measures for diagnosing other sleep disorders. Diagnostic Grading: A2	The SLEEP-50 Questionnaire is a valid psychometric tool, designed to detect many sleep disorders with 8 out of 50 items pertaining to insomnia. Its length limits its use for population studies which focus only on insomnia.
Kessler et al. (2010)	Case-control; psychometric instrument development (Brief Insomnia Questionnaire—BIQ)	83 INS and 120 HS, derived from the America Insomnia Survey’s 10.094 individuals.	BIQ; CΒDI	Sens 72.6%; Spec 98.9%; PPV 96.7%; NPV 88.7%; ROC-AUC = 0.86***	The reliability and validity of BIQ are very satisfactory. Diagnostic Grading: A1	The BIQ is quite lengthy and somewhat difficult to rate but its sophisticated design makes it a valuable tool, particularly for large epidemiological studies.
(b) Rating scales and questionnaires for assessing beliefs and attitudes about sleep
Smith and Trinder (2001)	Case-control	19 INS; 19 HC	DBAS; CΒDI; SII; SDQ; SWAI	ROC-AUC = 0.92; Sens 89%, Spec 78% (for cut-off 34.9)	Validity measures were quite satisfactory for DBAS; similar discrimination accuracy was also shown by SII and a psychiatric subscale of SDQ. Diagnostic Grading: A1	DBAS showed high accuracy in discriminating between INS and HC.
Harvey and Espie (2004)	Case-control; psychometric instrument validation (Glasgow Content of Thoughts Inventory—GCTI)	29 INS; 29 HS	GCTI	Sens 100%; Spec 83%; α = 0.87; T-R = 0.88 (3 weeks); Ext val: r = 0.65 to a sleep diary, r = 0.48 to actigraphy.	The GCTI is a scale for reporting pre-sleep thought content; it has been found to distinguish satisfactorily between insomniacs and good sleepers. External validity to actigraphy was quite low, in contrast to the same measure vs. a sleep diary. Diagnostic Grading: A1	The GCTI scale is intended to be used mainly in the field of cognitive psychology, focussing on the assessment of pre-sleep cognitive activity, and it is not designed to measure insomnia per se; thus it seems to have a rather limited usefulness for wide application in every-day clinical practice. The stronger correlation of the GCTI to the sleep diary than to actigraphy highlights the subjective nature of insomnia.
Broomfield and Espie (2005)	Case-control; psychometric Instrument development (Glasgow Sleep Effort Scale—GSES)	89 INS;102 HS	GSES; CΒDI; PSQI; HADS; DBAS	Sens 93%; Spec 87%; PPV 87%; NPV 94%; α = 0.77; Ext val: high to PSQI.	The GSES can distinguish satisfactorily between insomniacs and good sleepers. Diagnostic Grading: A1	The GSES scale has not been developed to measure the condition insomnia per se, but it is rather devised as a measure of the voluntary attempt to control sleep which leads to psychophysiological insomnia.
Carney et al. (2010)	Case-control	Four INS groups (329 PI, 76 PBUI, 114 IMC, 530 CIMS); 335 HC	DBAS-16; CΒDI; sleep log	α = 0.82; for all groups of insomniacs: ROC-AUC = 0.86 Sens 80% Spec 76% (for cut-off 3.8); for PI only: AUC = 0.78 Sens 66% Spec 76%.	DBAS-16 shows a moderately high discrimination accuracy and good sensitivity and specificity. A slightly lower cut-off (3.5) may be useful in highly screened INS patients (e.g. PI and IMC) to increase the sensitivity of the measure. Diagnostic Grading: A2	DBAS-16 is a reliable and valid tool for use across a range of insomnia patient groups.
Crönlein et al. (2014)	Case-control	34 INS; 30 SAS; 31 RLS; 26 SAS + RLS; 24 HYP; 84 HC	DBAS-16; CΒDI; RIS; PSQI; PSG	DBAS-16 (92.2 ± 22.8 INS vs. 56.5 ± 25.5 HC, d = 1.47, p < 0.001) Ext val: r = 0.603 to RIS; r = 0.346, −0.300, 0.470 (to subjective SOL, TST, WASO, resp.); r = −0.218, 0.238, −0.253, −0.218 (to PSG determined TST, WASO, N2%, SE%, respectively).	DBAS-16 score was moderately correlated with RIS in the whole sample; it showed low correlation with several subjective sleep measures and with PSG variables in patients. Diagnostic Grading: B2	Increased DBAS scores were found not only in primary insomnia, but also in other sleep disorders.
Hertenstein et al. (2015)	Case-control	47 INS; 52 GSC	DBAS-16; CΒDI; GSES; ISI; PSG; sleep log	DBAS-16 (4.9 ± 1.8 INS vs. 2.3 ± 1.1 GSC, d = 1.76) GSES (6.7 ± 3.2 INS vs. 1.0 ± 1.4 GSC, d = 2.31) Ext val: β = −0.41 to PSG-determined total sleep time Correlation of ISI to GSES r = 0.35 and to DBAS r = 0.32 (both p < 0.05).	DBAS-16 score was negatively related to PSG-determined total sleep time with a medium effect size. Diagnostic Grading: B1	DBAS was not related to ISI in this study.
(c) Inventories for the assessment of personality patterns
Kales et al. (1983)	Case-control	279 INS; 97 GS	Minnesota Multiphasic Personality Inventory (MMPI); CΒDI	MMPI clinical scales^**** differed between INS and GS as follows (mean ± SD, Cohen’s d): D (71.6 ± 10.8 vs. 52.8 ± 16.7, d = 1.22), Pt (67.7 ± 8.9 vs. 53.7 ± 13.4, d = 1.13), Hy (66.8 ± 7.9 vs. 54.8 ± 11.7, d = 1.11), Hs (63.2 ± 7.9 vs. 50.2 ± 13.4, d = 1.07), Sc (66.4 ± 9.8 vs. 54.5 ± 15.0, d = 0.86), Pd (65.0 ± 10.8 vs. 56.2 ± 11.7, d = 0.77), Pa (60.7 ± 8.9 vs. 55.4 ± 10.0, d = 0.54), Ma (57.2 ± 9.8 vs. 55.0 ± 11.7, d = 0.20). At least one pathologically elevated scale in 76% of INS vs. 29% of GS (Sens 76%, Spec 88.3%); mean number of elevated scales per subject 2.6 in INS vs. 0.5 in GS.	Pathologically elevated MMPI scales in INS; profiles of INS were consistent with the presence of neurotic depression, rumination, chronic anxiety and inhibition of emotions, in particular anger. Diagnostic Grading: A2	Predominant personality style in insomnia is indicative of internalisation of negative emotions and other psychological disturbances; this pattern leads to psychophysiological activation and to the vicious cycle of generation and perpetuation of insomnia.
Stephan et al. (2018)	Longitudinal	4 general population samples; N > 22.000	Midlife Development Inventory; Big Five Inventory; self-administered questionnaire to measure sleep quality, follow-up after a 4–12 year period	Correlation coefficients (based on random effect metaanalysis) were generally low: impaired sleep quality at baseline with neuroticism (0.23) and extraversion (−0.06); decline in sleep quality at follow-up with neuroticism (0.10), extraversion (−0.03) and conscientiousness (−0.02).	Low neuroticism and high extraversion were related to better sleep quality at baseline and over time, and low conscientiousness was associated with a decrease of sleep quality over time, although the strength of the associations was weak. Diagnostic Grading: C4	Longitudinal relations were found between personality traits and poor and worsening sleep quality.
Van de Laar et al. (2010)	Review	38 articles on insomnia and personality; MMPI: 8 studies with a total of 857 INS	PubMed Search for MMPI in INS	Four MMPI clinical scales were pathologically elevated in INS: D (8/8 studies), Hy (7/8), Hs (6/8), Pt (6/8); two scales less frequently found to be elevated were Pd (2/8), Sc (1/8); no elevation for Mf, Pa, Ma, Si.	Neuroticism, internalisation, anxiety and perfectionism are commonly associated with insomnia. Diagnostic Grading: C2	Further longitudinal studies are needed to explore the relationship between personality and insomnia, particularly in relation to whether the persistence of chronic insomnia leads to changes in personality.

α: Cronbach’s alpha; AIS: Athens Insomnia Scale; β: regression coefficient beta; BIQ: Brief Insomnia Questionnaire; CBDI: clinically-based diagnosis of insomnia; CIMS: complains of initiating maintaining sleep; d: Cohen’s d; DBAS: Dysfunctional Beliefs and Attitudes about Sleep scale; DBAS-10: 10-item version of DBAS; DBAS-16: 16-item version of DBAS; DSM-5: Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition; Ext val: external validity; g: Hedge’s g; GCTI: Glasgow Control of Thoughts inventory; GS: good sleepers; GSAQ: Global Sleep Assessment Questionnaire; GSC: good sleeper controls; GSES: Glasgow Sleep Effort Scale; HADS: Hospital Anxiety and Depression Scale; HC: healthy controls; HYP: hypersomnia; HS: healthy subjects; ICD-10: International Classification of Diseases,10th Revision; IMC: insomnia and medical condition; INS: insomniacs; ISI: Insomnia Severity Index; k: Cohen’s kappa; MMPI: Minnesota Multiphasic Personality Inventory; MOS-SPI: Medical Outcome Study Sleep Problems Index I; N2: non-rapid eye movement sleep, stage 2; NPV: negative predictive value; OSA: obstructive sleep apnoea; OSD: other sleep disorders patients; PBUI: prolonged benzodiazepine users for insomnia; PI: primary insomniacs; PPV: positive predictive value; PSG: polysomnography; PSQI: Pittsburgh Sleep Quality Index; Psy: psychiatric patients; pts: patients; RIS: Regensburg Insomnia Scale; RLS: restless leg syndrome; ROC-AUC: receiver operating characteristic-area under the curve; SAS: sleep apnoea syndrome; SDQ: Sleep Disorders Questionnaire; SE: sleep efficiency; Sens: sensitivity; SF-36: Short Form Health Survey; SII: Sleep Impairment Index; SOL: sleep onset latency; sl lat: sleep latency; Spec: specificity; SPS: Sleep Problems Scale; SRBD: sleep related breathing disorders; SWAI: Sleep-Wake Activity Inventory; TST: total sleep time; T-R: test-retest reliability (week interval in brackets); WASO: wake after sleep onset.

Primary measure(s) assessed in each study are highlighted by the use of bold characters in the Instruments and Methods column.

*All values presented on this table pertain only to the sample of insomniacs of each study, unless otherwise specified.

**As shown by Pallesen et al. (2008).

***The psychometric characteristics of the BIQ provided in the paper are examined based on various diagnostic criteria (DSM -IV-TR, ICD-10, RDC); herewith we report the values pertaining to the diagnosis of insomnia when any of the above criteria are met.

****MMPI clinical scales: Hs: hypochondriasis; D: depression; Hy: hysteria; Pd: psychopathic deviate; Mf: masculinity/femininity; Pa: paranoia; Pt: psychasthenia; Sc: schizophrenia; Ma: hypomania, Si: social introversion.

Table 3. Studies of biological measurements in diagnosing insomnia.

Citation	Type of study	Type and number of subjects	Instruments and methods	Results	Main conclusions	Comments
(a) Polysomnography and sleep EEG analysis
Buysse et al. (2008)	Case-control	48 PI (29 women), 25 GSC (15 women)	PSG power spectral analysis (1–50 Hz) of EEG in NREM sleep with central EEG leads referenced to A1 + A2	No group difference in EEG power for whole-night NREM sleep; PI had higher power than GSC in 18–40 Hz in the first NREMP (p < 0.05); PI women vs. GSC women had higher power in 16–44 Hz (first three NREMPs) and in 3–6 Hz (all NREMPs) (p < 0.05); PI men vs. GSC men showed no power differences.	Women with PI showed increased high-frequency and low-frequency EEG power in NREM sleep compared to controls, particularly in early NREMPs; men with PI showed no EEG power differences vs. controls. Diagnostic Grading: D	Sex and NREMP rank may affect quantitative EEG differences between PI and controls; hyper-arousal and ‘deep’ sleep may coexist in women with PI.
Chouvarda et al. (2012)	Case-control	10 PI, 11 GSC	CAP rate based on PSG	Diagnostic potential of increased CAP rate: Sens 81.8% Spec 100% Diag Acc 90.5%.	Increase in CAP rate differentiates INS from GSC. Diagnostic Grading: A1	Increased CAP reflects sleep instability; promising tool in the diagnosis of insomnia; proof for the hyperarousal concept of insomnia.
Spiegelhalder et al. (2012)	Case-control	25 PI, 29 GSC	PSG power spectral analysis	Differences between power EEG frequency spectra in PI vs. GSC regarding wave bands: sigma (12–16 Hz; 1.89 ± 0.46 vs. 1.49 ± 0.43, d = 0.90), beta 1 (16–24 Hz; 0.51 ± 0.50 vs. 0.19 ± 0.36, d = 0.74), beta 2 (24–32 Hz; −0.63 ± 0.45 vs. −0.93 ± 0.39, d = 0.72).	Spectral power values for beta and sigma frequency bands are elevated during NREM in PI. Diagnostic Grading: B3	Spectral analysis data reflect a marker of cortical arousal; proof for hyperarousal theory.
Baglioni, Regen, et al. (2014)	Meta-analysis of 23 PSG studies	582 PI, 485 GSC	PSG; self-ratings	Based on studies with subjectively defined insomnia, PI differed from GSC regarding sleep continuity measures: SE (d = −0.7), TST (d = −0.6), SOL (d = 0.4), NA (d = 0.6), and WASO (d = 0.6). Also they differed in terms of duration of SWS (d = −0.8) and REM (d = −0.6).	Disturbed sleep continuity and reduced SWS and REM in PI. Diagnostic Grading: B3	Significant differences of PSG measures between PI and GSC (for TST about 25 min). Yet, subjective TST difference is much larger (about 2 h).
Riedner et al. (2016)	Case-control	8 CI, 8 GSC (sex and age-matched)	Sleep diaries; single-night PSG with 256-channel high-density EEG recording power spectral analysis and sLORETA (for cortical source imaging) of NREM sleep	CI vs. GSC had more power in high-frequency EEG activity (>16 Hz: for beta p < 0.02, for low gamma p < 0.004), widespread across the scalp; more power in alpha EEG activity (8–12 Hz, p < 0.03), even in sleep stage N3 (p < 0.04); in source imaging analysis CI vs. GSC showed elevated alpha EEG activity during sleep stage N3 in sensory and sensorimotor cortical areas (p < 0.06).	Possible hyperarousal, even in the deepest stage of sleep, in chronic insomnia subjects vs. controls. Sensory and sensorimotor cortical areas may be relatively more active, even in sleep stage 3, in CI vs. GSC. Diagnostic Grading: D	Possible sleep dysregulation in insomnia, denoting hyperarousal, seems to be localised in certain brain areas.
Svetnik et al. (2017)	Case-control	803 INS (509 women), 811 GSC (502 women)	Single-night PSG power spectral analysis (1–32 Hz) of NREM sleep EEG data, for each of three equal (one-third) parts of the night	INS vs. GSC under 40–45 years old had reduced power in the delta band (1–4 Hz) during Part 1 of the night (p < 0.01); male INS under 40 years old had reduced power in the theta band in Part 1 of the night (p < 0.05); female INS had increased power in the beta2 band (25–32 Hz) in all parts of the night (ratio INS/GSC power from 1.11 to 1.16; p < 0.05); male INS had reduced power in the alpha band (9–12 Hz) during all parts of the night (ratio INS/GSC power from 0.82 to 0.92; p < 0.01).	Evidence for hyperarousal in INS, based on EEG spectral analysis; there may be an influence of age, sex and part of the night. Diagnostic Grading: D	Sleep hyperarousal in insomnia, possibly more pronounced among female subjects.
(b) Actigraphy
Natale et al. (2009)	Case-control	126 INS, 282 NS	ACT (Mini-Motionlogger) for ≥7 consecutive nights (mean 8.4 nights)	Diagnostic potential of ACT: (a) SOL (12 min cut-off): ROC-AUC 0.68, Sens 0.55, Spec 0.81, PPV 0.56, NPV 0.80; (b) TST (440 min cut-off): ROC-AUC 0.68, Sens 0.66, Spec 0.61, PPV 0.43, NPV 0.80; (c) WASO (25 min. cut-off): ROC-AUC 0.75, Sens 0.66, Spec 0.75, PPV 0.54, NPV 0.83; (d) SE (92% cut-off): ROC-AUC 0.83, Sens 0.71, Spec 0.78, PPV 0.58, NPV 0.86. Discriminant score (based on combination of actigraphy-based TST, SOL and NA > 5): ROC-AUC 0.87, Sens 0.64, Spec 0.92, PPV 0.81, NPV 0.83	Actigraphy-based sleep parameters (each alone or in combination) differentiated INS from NS. Diagnostic Grading: A1	One of the few large studies providing quantitative criteria for identifying INS using Mini-Motionlogger
Natale et al. (2009)	Case-control	151 INS, 342 HC	ACT (Actiwatch 64) for one week	Diagnostic accuracy: (a) SOL (14 min cut-off): ROC-AUC 0.64, Sens 0.42, Spec 0.82, PPV 0.50, NPV 0.76; (b) WASO (40 min cut-off): ROC-AUC 0.74, Sens 0.68, Spec 0.55, PPV 0.40, NPV 0.79; (c) SE (87% cut-off): ROC-AUC 0.72, Sens 0.50, Spec 0.74, PPV 0.47, NPV 0.77.	ACT can differentiate INS from NS. Diagnostic Grading: A2	One of the few large studies providing quantitative criteria for identifying INS using Actiwatch 64. The quantitative criteria were not the same as those obtained by the same group with a different device denoting the need to adopt shared technical solutions.
(c) Heart rate (HR) and heart rate variability (HRV)
Bonnet and Arand (1998)	Case-control	12 INS, 12 HC	ECG for HR; PSG over a 36-h sleep laboratory stay	Heart period (beat-to-beat interval) and its SD were lower in all stages of sleep in insomniacs vs. HC (p < 0.001 for both comparisons). Heart period in SWS was shorter in INS vs. HC (0.84 vs. 1.095, p = 0.02, d = −0.91–1.19*).	HR is increased and its variability (HRV) is decreased in INS vs. HC. Diagnostic Grading for HR: B2	HR and HRV changes indicate increased sympathetic nervous system activity in insomniacs during sleep.
Fang et al. (2008)	Case-control	18 INS, 21 NS	Resting HRV under paced breathing (resting wakefulness); neuropsychological evaluation.	No statistically significant differences between groups for HRV. Trend for lower resting HRV in INS vs. NS (HF of HRV 6.31 ± 0.93 in INS, vs. 6.91 ± 1.15 in NS; d = −0.57).	Heart rate variability may be decreased in insomniacs even during wakefulness. Diagnostic Grading for HRV: B4	Weak evidence of autonomic system dysregulation in insomnia.
Spiegelhalder et al. (2011)	Case-control	58 INS, 46 HC	HRV assessments in PSG-derived 5-min periods during waketime, stage 2 NREM, and REM.	INS vs. HC: lower wake-to-sleep HR reduction and lower variability of R-R intervals. Nocturnal HF of HRV (as indicator of parasympathetic activity) was in INS-SSD vs. INS-NSD vs. HC 522 ± 116 vs. 902 ± 224 vs. 1054 ± 179 for NREM and 497 ± 121 vs. 684 ± 169 vs. 1002 ± 203 for REM (values in ms^2 ± SEM); d-values for INS-SSD vs. HC, INS-SSD vs. HC and INS-SSD vs. INS-NSD were respectively, −0.52, −0.13, −0.40 for NREM and −0.44, −0.26, −0.24 for REM sleep.	Based on HF of HRV values, INS with SSD showed the lowest parasympathetic activity, HC the highest; INS without SSD had values between the above two groups. Diagnostic Grading for HRV: B4	Alterations in HRV associated with sympathovagal disbalance may depend on sleep duration in insomniacs.
de Zambotti et al. (2013)	Case-control	9 INS, 9 NS	Assessment of impedance cardiography for HR and HRV during a night of PSG; PSQI; AIS	(a) HR in bpm was 71.10 ± 7.24 in INS vs. 61.92 ± 12.01 in NS during time awake (g = 0.88); the difference between stage awake and sleep stages was much smaller in NS vs. INS (group by stage interaction p < 0.001). (b) HRV spectral analysis: reduction from wake to sleep of LF/HF ratio and increase in HF; no difference between groups. (c) PEP (inversely related to β-adrenergic activity) was shorter in INS vs. NS (p < 0.05). (d) PEP was correlated with AIS (r = 0.53) and PSQI (r = 0.58); both p < 0.05.	(a) Strong reduction in HR from wake to sleep in INS. (b) No differences between groups in spectral analysis findings. (c) Constant hyperactivation of the sympathetic nervous system throughout the whole night in INS vs. NS. (d) Correlation between high physiological sympathetic activation and low score in subjective sleep assessment measurements. Diagnostic Grading for HR: B3	Sympathetic hyper-activation is a feature of INS.
Farina et al. (2014)	Case-control	85 INS, 55 HC	HR based on home-based PSG; subjective sleep evaluation	HR (in bpm) in INS vs. HC was increased in wake before sleep (67.8 ± 8.7 vs. 59.0 ± 7.3; p < 0.001, d = 1.08), in early stage N2 sleep (64.8 ± 7.6 vs. 49.0 ± 5.4; p < 0.001, d = 2.32) and late-stage N2 sleep (60.9 ± 8.0 vs. 53.5 ± 10.4; p < 0.001, d = 0.82); no significant differences for stages N3, REM and wake state after final awaking. HRV was lower in INS vs. HC across periods of wakefulness and the various sleep stages.	Modifications of heart rate and HRV were consistent with increased sympathetic activity while being awake before sleep and in Stage-2 NREM sleep; no differences during slow-wave sleep, REM sleep and post-sleep awake. Diagnostic Grading for HR: B1	Autonomic hyperarousal is a feature of insomnia, both before and during sleep.
(d) Skin conductance
Broman and Hetta (1994)	Case-control	40 INS (primary or secondary to psychiatric disorders), 20 HC	Electrodermal activity (EDA) during a habituation paradigm; PSG	(a) SCL was higher in INS vs. HC in all three conditions (pre-, during and post-stimulation) (p < 0.005 vs. HC) (b) Non-habituation of EDA with repeated stimuli 16/40 INS vs. 2/17 HC (χ^2 = 10.5, df = 2, p < 0.01) Sens 40% Spec 88.9%; non-habituation was associated with higher EDA (p < 0.001) and shorter sleep duration (subjectively and objectively assessed; p < 0.005 for both).	Excessive daytime arousal, as reflected by EDA, is associated with persistent insomnia. Diagnostic Grading for non-habituation of EDA: A4	Proof of daytime arousal in insomnia.
(e) Thermoregulation
Gradisar et al. (2006)	Case-control	11 INS, 8 NS	Core body (Tc) and finger (Tf) temperature in 26-h constant routine	Greater increases in Tf 5 min after lights out in INS vs. NS (1.5 ± 1.2 °C vs. 1.2 ± 1.2 °C, p < 0.001, g = 0.24). Higher mesor of Tc in INS vs. NS (37.0 ± 0.2 °C vs. 36.8 ± 0.2 °C, p = 0.03, g = 0.96).	INS have grater finger temperature increases when attempting to sleep, and higher core body temperature. Diagnostic Grading for Tc: B3	Insomnia may be a disorder of thermoregulation with impaired heat loss.
(f) Oxygen consumption rate
Bonnet and Arand (1996)	Case-control	10 INS, 10 HC	2 days PSG; VO2 consumption; MSLT; MMPI; sleep history	Mean VO2 consumption rate during the whole 24 h period was 296 vs. 266 ml/min (INS vs. HC, p < 0.01, g = 0.95). Mean VO2 consumption during the sleep period was 280 vs. 256 ml/min (INS vs. HC, p < 0.0001, g = 0.76).	Metabolic rate (as reflected in VO2 consumption rate) was elevated in INS vs. HC across the day and throughout the night. Diagnostic Grading: B3	Insomnia is a state of hyperarousal during the whole 24 h period.
Bonnet and Arand (1998)	Case-control	9 INS with subjective complaint only (relatively normal sleep on their PSG), 9 HC.	2 days PSG; VO2 consumption; MSLT; MMPI; sleep history	Mean VO2 consumption rate during the whole 24 h period was 304 ± 26 vs. 286 ± 34 ml/min (INS vs. HC, p < 0.001, g = 0.57). Mean VO2 consumption rate during the sleep period did not differ between the two groups.	Increases in VO2 in patients with Sleep State Misperception Insomnia (SSMI) were found only during daytime. Diagnostic Grading: B4	Daytime metabolic rate is increased in insomnia, even in sleep state misperception insomnia.
(g) Neuroimaging
Smith et al. (2002)	Case-control	5 PI, 4 GSC	SPECT; PSG	Cerebral blood flow is reduced in PI vs. GSC during NREM for: FMC (212.7 ± 42.6 vs. 271.4 ± 26.3, p < 0.05, g = −1.43), OC (190.9 ± 20.5 vs. 239.6 ± 20.5, p < 0.03, g = −2.11), BG (157.4 ± 21.8 vs. 233.1 ± 36.8, p < 0.006, g = −2.31), PC (182.1 ± 27.1 vs. 232.6 ± 27.6, p < 0.03, g = −1.64).	Cerebral blood flow appeared to be reduced during NREM in primary insomnia. Diagnostic Grading: B1	Small sample size; finding seems to be contrary to the findings of other neuroimaging studies, making its high diagnostic validity questionable.
Altena et al. (2008)	Case-control	21 INS, 12 GSC	fMRI; verbal fluency tests	INS vs. GSC showed less activation in the LMPC (z = 3.73–4.18; d = 1.61–1.82) and LIFG (z = 3.30–4.17; d = 1.44–1.82) during fluency tests; these findings were partially restored after non-pharmacological intervention for treating insomnia.	Reduced task-related frontal activity in insomniacs, reversed after treatment. Diagnostic Grading: B1	The reduced frontal activity in cognitive tasks may be associated with persistent worry and preoccupation in insomniacs.
Kay et al. (2016)	Case-control	44 PI, 40 GSC	PET; PSG	Decline in regional cerebral metabolic rate for glucose from wakefulness to sleep was lower in PI vs. GSC in the following cortical areas: praecuneus/posterior cingulate, anterior cingulate/medial frontal, insula, anterior temporal (all p < 0.05).	Decrease of brain metabolism from wake to NREM is less pronounced in PI compared to GSC. Diagnostic Grading: D	Insomnia may be related to a failure of arousal mechanisms to decline in activity from wake to sleep.
Regen et al. (2016)	Case-control	20 PI, 20 GSC	fMRI; PSG	Waking DMN connectivity was correlated negatively with SE (hip-ACC r^2 = 0.24; hip-PCC r^2 = 0.28; hip-vmPFC r^2 = 0.26, RSC-vmPFC r^2 = 0.24) and positively with SOL (RSC-ACC r^2 = 0.26; RSC-PCC r^2 = 0.17; RSC-vmPFC r^2 = 0.31). No statistically significant group differences between PI and GSC.	No group differences in functional connectivity between primary insomniacs and good sleepers. Diagnostic Grading: C3	DMN connectivity was found to be correlated to some PSG indices; no differences were found between insomnia and controls.
Leerssen et al. (2019)	Case-control	65 INS, 65 GSC	fMRI; ISI; sleep diary	Stronger functional connectivity in INS vs. GSC between bilateral hippocampus and a cluster of voxels in the left middle frontal gyrus (Zmax = 4.22; p = 0.035 in cluster based correction for multiple comparisons, d = 1.05); the strength of this connectivity was associated with insomnia severity and sleep quality (r = 0.37 and r = −0.31, respectively; p < 0.01 for both).	In insomnia there is increased hippocampus-prefrontal connectivity; this increase is correlated to subjective measures of insomnia severity and poor sleep quality. Diagnostic Grading: B1	A part of the circuit activating with maladaptive rumination and deactivating with sleep shows higher functional connectivity in insomnia.
(h) HPA axis
Vgontzas et al. (2001)	Case-control	11 INS, 13 HC	Serial 24h-plasma samplings of ACTH and cortisol	INS vs. HC: 24h-ACTH 4.2 ± 0.3 vs. 3.3 ± 0.3 pM, p = 0.04, d = 3.0; 24 h-cortisol 218.0 ± 11.0 vs. 190.4 ± 8.3 nM, p = 0.07, d = 2.87. Higher values in INS, most prominent in the evening and first half of the night. Circadian rhythm retained. Degree of cortisol elevation correlates with degree of objective shortening of sleep duration.	HPA-axis activity elevated in INS vs. HC, especially in the evenings and first half of the night, related to objectively increased sleep fragmentation. Diagnostic Grading: B1	HPA-axis findings are more prominent in insomnia with objectively verified short sleep duration.
Riemann et al. (2002)	Case-control	10 PI, 10 HC	PSG; evening and nocturnal serum cortisol and melatonin sampling (from 19:00 to 09:00 h in 30 min intervals)	Differences of PSG sleep continuity and sleep architecture variables between PI and HC were negligible. Cortisol levels were not different between PI and HC: AUC values of cortisol levels (in μg/dl × 30 min) were for PI 214.4 ± 62.5 and for HC 253.7 ± 75.9, p = 0.245, g = −0.54.	In PI with minor alterations of objective sleep, serum cortisol is not affected. Diagnostic Grading: B4	Involvement of HPA axis seems to depend on degree of objective sleep disturbance.
Seelig et al. (2013)	Case-control	13 PI, 12 HC (all women)	PSG; midnight and early morning salivary cortisol	Increased mid-night salivary cortisol higher in PI vs. HC (1.4 ± 1.4 mmol/lt vs. 0.7 ± 0.4 mmol/lt, d = 0.67, p = 0.02); no significant difference for morning salivary cortisol (10.3 ± 6.3 mmol/lt vs. 7.3 ± 3.5 mmol/lt, p = 0.18, d = 0.58).	Mid-night salivary cortisol levels are increased in primary insomnia. Diagnostic Grading: B4	HPA-activity at the first half of the night seems to be more reliable in insomnia than morning cortisol.
Mikoteit et al. (2019)	Case-control	60 INS, 30 HC	ISI; morning salivary cortisol.	Insomnia is associated with decreased cortisol awakening reaction (CAR, but the association is rather weak; g = 0.53–0.59); ISI correlates inversely with CAR.	Insomnia is not related to increased morning salivary cortisol but rather to decreased CAR. Diagnostic Grading: B4	The deficit of cortisol awakening reaction in insomniacs seems to be a characteristic of insomnia, rather than an increase of morning cortisol levels.
(i) Melatonin
Hajak et al. (1995)	Case-control	10 PI, 5 HC	PSG; hourly sampling of nocturnal plasma melatonin concentrations	Melatonin peak at the middle of the night was lower in PI (82.5 ± 26.5 pg/ml) vs. HC (116.8 ± 13.5 pg/ml; g = −1.39, p < 0.01), especially in chronic patients (72.1 ± 25.0 vs. 98.2 ± 24.9 pg/ml in patients with PI duration more than 5 vs. <5 years, g = −0.94); g-values for chronic PI vs. HC and for non-chronic PI vs. HC were −1.97 and −0.86, respectively. Plasma melatonin levels begin increasing earlier in the evening in PI vs. HC.	Earlier onset and lower peak of melatonin typical for chronic primary insomnia. Diagnostic Grading: B2	Circadian rhythm of melatonin secretion is disturbed in patients with chronic primary insomnia; proof-of-concept study.
Riemann et al. (2002)	Case-control	10 PI, 10 HC	PSG; evening and nocturnal serum cortisol and melatonin sampling (from 19:00 to 09:00 h in 30 min intervals)	AUC values of melatonin levels (in pg/ml × 60 min) were lower for PI (515.5 ± 174.6) than for HC (682.8 ± 124.2), p = 0.029, g = −1.06; the main difference occurred during the first half of the night. The effect of objective measures (PSG) of insomnia was negligible.	Insomnia, even with no objective sleep changes, is related to a deficit in nocturnal melatonin, especially during the first half of the night. Diagnostic Grading: B2	Circadian sleep regulation is affected in insomnia.
(j) Marker of inflammation: CRP
Parthasarathy et al. (2015)	Longitudinal, community-based	1409 adults (including 249 intermittent and 128 persistent insomniacs)	CRP; assessment for presence of insomnia at two time points with an interval of 6 years; 20 year follow up, for assessment of mortality rate.	Participants with insomnia at both time-points (6 years apart) were more likely to die during the 20-year follow-up period than non-insomniacs (Hazard Ratio: 1.58, 95%CI: 1.02–2.45); no significant difference for those complaining of insomnia at either time-point (Hazard Ratio:1.22, 95%CI: 0.86–1.74). Adjusted mortality Hazard Ratio did not change when CRP was included in the model (1.93 vs. 1.97). Geometric means of CRP measurements at the second time-point (but not at baseline) for subjects with insomnia at both time-points vs. insomnia at either time point vs. non-insomniacs were 2.86 vs. 1.61 vs. 1.52 mg/lt, respectively (ANOVA p = 0.02).	Increase of CRP is associated with persistent insomnia but does not seem to be an important mediator between insomnia and mortality. Diagnostic Grading: D	Persistent insomnia relates to increased mortality risk, independent of systematic inflammation.
(k) Marker of neuroplasticity: BDNF
Mikoteit et al. (2019)	Case-control	60 INS, 30 HC	ISI; PSG; serum BDNF	Decreased BDNF separates INS from HC (Hedge’s g = 1.75), ROC-AUC 0.88, Sens 0.78, Spec 0.79, PPV 0.88, NPV 0.66. Low BDNF correlated with decreased REM sleep, but not with any objective sleep continuity parameters in the PSG.	Decreased BDNF is associated with subjective sleep difficulty, but not for objectively assessed poor sleep continuity. Diagnostic Grading: A1	BDNF appears to be a biomarker for the clinical diagnosis of insomnia. A possible link between BDNF and insomnia seems to be via regulation of REM sleep
Fan et al. (2019)	Case-control	57 INS: 30 with TST < 6 h (short sleep duration insomniacs, INS-SSD) and 27 with TST ≥ 6 h (normal sleep duration insomniacs, INS-NSD), 29 HC	Clinical diagnosis for insomnia; PSG; neuropsychological tests; serum BDNF	BDNF levels in INS-SSD vs. INS-NSD vs. HC were 11.4 ± 0.3 vs. 17.4 ± 0.3 vs. 20.5 ± 1.2; d-values for INS-SSD vs. HC, INS-NSD vs. HC and INS-SSD vs. INS-NSD were −10.49, −3.49, and −20.00, respectively. In INS-SSD, BDNF levels correlated with test scores of spatial span (r = 0.42), visuospatial memory (r = 0.50) and continuous performance (r = 0.42).	INS-SSD had lower BDNF than INS-NSD and HC. INS-NSD had lower BDNF than HC. Cognitive performance correlated with BDNF levels in INS-SSD. Diagnostic Grading: B1	Low BDNF differentiates INS from HC and it may be a mediator between insomnia and cognitive impairment in insomniacs with short sleep duration.

95%CI: 95% confidence interval; ACC: anterior cingulate cortex; ACT: actigraphy; ACTH: adrenocorticotrophin hormone; AIS: Athens Insomnia Scale; AUC: area under the curve; BDNF: brain-derived neurotrophic factor; BG: basal ganglia; CAR: cortisol awakening reaction; CAP: cyclic alternating pattern; CI: chronic insomniacs; CRP: C-reactive protein; DMN: default mode network; EDA: electrodermal activity; EEG: electroencephalography; FMC: frontal medial cortex; fMRI: functional magnetic resonance imaging; g: Hedge’s g; GSC: good sleeper controls; HC: healthy controls; HF: high frequency; hip: hippocampus; HPA: hypothalamus-pituitary-adrenal; HR: heart rate; HRV: heart rate variability; Hz: hertz; ID: Insomnia Disorder; IL-6: interleukin-6; INS: insomniacs; ISI: Insomnia Severity Index; LF: low frequency; LIFG: left inferior frontal gyrus; LMPC: left medial pre-frontal cortex; MMPI: Minnesota Multiphasic Personality Inventory; MSLT: multiple sleep latency test; NA: number of awakenings; NA > 5: number of awakenings longer than 5 min; NPV: negative predictive value; NREM: non-rapid eye movement; NREMP: non-REM period; NS: normal sleepers; NSD: normal sleep duration; OC: occipital cortex; PC: parietal cortex; PCC: posterior singulate cortex; PEP: pre-ejection period; PET: positron emission tomography; PI: primary insomniacs; PPV: positive predictive value; PSG: polysomography; PSQI: Pittsburgh Sleep Quality Index; RCT: randomised controlled trial; REM: rapid eye movement; ROC-AUC: receiver operating characteristic-area under the curve; RSC: retrosplenial cortex; SCL: skin conductance level; SD: standard deviation; SE: sleep efficiency; SEM: standard error of the mean; Sens: sensitivity; SL: sleep latency; sLORETA: standardised low-resolution brain electromagnetic tomography analysis; SOI: sleep onset insomnia; SOL: sleep onset latency; Spec: specificity; SPECT: single photon emission computerised tomography; SSD: short sleep duration; SWS: slow wave sleep; Tc: core body temperature; Tf: finger temperature; TIB: time in bed; TNF-α: tumour necrosis factor-a; TST: total sleep time; TWT: total wake time; vmPFC: ventromedical pre-frontal cortex; VO₂: volume of oxygen; WASO: wake after sleep onset.

Primary measure(s) assessed in each study are highlighted by the use of bold characters in the Instruments and Methods column.

*Range of d-values based on a range of SD values in different populations, as reported in the study by O’Neil et al. (2016).

Table 4. Overview of the diagnostic potential for insomnia of psychometric tools and biological measurements.

Diagnostic potential	Psychometric tools	Biomarkers
Excellent^a	Scales for assessing insomnia Scales for assessing beliefs and attitudes about sleep	Actigraphy* BDNF CAP**
Moderate^b	MMPI	HR around sleep onset Neuroimaging***
Limited^c		Melatonin
Inconsistent^d		HPA axis

BDNF: brain-derived neurotrophic factor; CAP: cyclic alternating pattern; HPA: hypothalamus-pituitary-adrenal; HR: heart rate; HR: heart rate variability; MMPI: Minnesota Multiphasic Personality Inventory.

*Absence of common standards of technical solutions and cut-off values.

**Based on only one study.

***Varying methodologies.

^aDiagnostic grading A1 in at least one study.

^bDiagnostic grading A2 or B1 in at least one study.

^cDiagnostic grading B2 in at least one study.

^dOne study providing positive evidence is outweighed by studies providing negative evidence.

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