Protocol at age of 46

A postal questionnaire on the participant’s health status and life style was conducted in 2012–2014. The questionnaires were mailed to all subjects whose addresses were known (n = 10,331). The response rate was 66% (n = 6,825). Subjects, who were living at known addresses in Finland, were invited to clinical examinations that were coordinated by the NFBC project center at the Center for Life Course Health Research (University of Oulu) with three laboratory units (Oulu, Southern and Northern Finland). A total of 5,861 (57%) subjects participated in clinical examinations between April 2012 and March 2014. The subjects entered the laboratory between 7:00 and 11:00 a.m. after an overnight fasting period (12 hours). Subjects were instructed to avoid smoking and drinking coffee during the examination day. Venous blood samples were drawn from an antecubital vein for the analysis of glycemic and lipid status. Serum glucose was analyzed using an enzymatic hexokinase/glucose-6-phosphate dehydrogenase method. Total cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol, and triglycerides were determined using an enzymatic assay method. The concentration of glycated and total hemoglobin were measured using immunochemical assay methods. The ratio is reported as percent hemoglobin A1c (NGSP). The samples were analyzed in NordLab Oulu, a testing laboratory (T113) accredited by Finnish Accreditation Service (FINAS) (EN ISO 15189) (All methods: Advia 1800; Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA). Systolic (SBP) and diastolic blood pressure (DBP) were measured three times in 1 minute periods (the two lowest systolic values and the corresponding diastolic values averaged) with an automated sphygmomanometer (Omron M10, Omron Healthcare, Kyoto, Japan) in a sitting position from the right arm after 15 minutes of rest. After various measurements, including the measurements of weight, height and circumferences of waist and hip, the participants had a light meal 60–90 min before the assessments of cardiovascular autonomic function. On a separate day after an overnight fasting period (12 hours), a 2-hour oral glucose tolerance test was conducted for these participants without medication for diabetes according to the recommendations [19].

The participant sat on a chair for instrumentation and the review of the protocol. A heart rate (HR) monitor (RS800CX, Polar Electro Oy, Kempele, Finland) was used to record R-R intervals (RRi) with an accuracy of 1 ms. In about half of the participants (Oulu laboratory unit only) spontaneous BRS was also assessed. Standard lead-II ECG (Cardiolife, Nihon Kohden, Tokyo, Japan), breathing frequency (MLT415/D, Nasal Temperature Probe, ADInstruments, Bella Vista, New South Wales, Australia), and blood pressure (BP) by finger plethysmography (Nexfin, BMEYE Medical Systems, Amsterdam, the Netherlands) were recorded during the protocol with a sampling frequency of 1,000 Hz (PowerLab 8/35, ADInstruments). The finger cuff was adjusted so that the values of SBP and DBP assessed by finger plethysmography (left arm) did not differ by more than 10 mmHg from those measured by automated sphygmomanometer at the same time (right arm, Omron M10). Physiological calibration of finger plethysmography was then turned off. An arm sling was used to support the left arm during the measurements. These procedures which were conducted in the seated position lasted for a total of 5–10 min which was followed by stabilization of HR for at least 1 min before the recording started. After 3-min recording in a seated position, the participant stood up and remained still in a standing position for 3 min while breathing spontaneously. The first 150 s of recording in seated position and the last 150 s in standing position were analyzed.

The RRi data were edited based on visual inspection (Hearts 1.2, University of Oulu, Oulu, Finland). Artefacts and ectopic beats were removed and replaced by local average. However, sequences with ≥10 consecutive beats of noise or ectopic beats were deleted. The RRi series with ≥80% accepted data were included in analyses. A total of 5,679 subjects attended RRi recordings of whom 5,473 (96%) had eligible HRV data for both phases of the protocol (seated and standing). Mean HR, root mean square of successive differences in RRi (rMSSD, ms), spectral power densities (fast Fourier transform, length 512 beats) at low (LF, 0.04–0.15 Hz, ms²) and high frequency (HF, 0.15–0.40 Hz, ms²) components of HRV, and their ratio (LF/HF) were analyzed [20]. Additionally, rMSSD was divided by 3^rd power of RRi (s) to remove RRi-dependency of rMSSD [21].

Continuous ECG, BP and respiration signals were imported into custom-made standalone Matlab-based software (Biosignal Processing Team, University of Oulu, Oulu, Finland). RRi and SBP values were extracted from the continuous ECG and BP recordings as discrete event series. Artefacts and ectopic beats were replaced using linear interpolation (<5% for accepted recording) and, thereafter, resampled at 2 Hz. Very-low-frequency components (< 0.04 Hz) were removed using the Savitzky-Golay method. A fast Fourier transform (Welch method, segments of 128 samples with 50% overlap, length 1024 samples) was performed to analyze the LF power of RRi and SBP oscillations (ms², mmHg²) for subsequent analysis of BRS by the alpha method if sufficient coherence (≥0.5) between LF oscillations in RRi and SBP was verified [22,23]. Out of 2,726 recordings, BRS was successfully calculated for 2,641 subjects in the seated position and 2,617 while they were standing. The main reasons why BRS could not be analyzed were numerous ectopic beats and technical artefacts.

Based on their answers in the postal questionnaire, subjects were defined as current smokers if they smoked regularly on ≥2 days/week. The amount of alcohol consumed per day was estimated from the questions measuring the frequency and the usual amount of beer, wine and spirits consumed on one occasion. The subjects were then categorized into two groups based on the highest sex-specific deciles rounded to the closest 10 g/d (cut-off for men: 40 g/d, women: 20 g/d). Total sitting time during waking hours was established by asking the subjects how many hours on average they sat on weekdays and the responses were dichotomized (cut-off 11 h/d). To evaluate the sufficiency of sleep, the subjects were asked how tired they felt in the morning during the first half an hour (very/somewhat tired = tired, somewhat/well rested = rested). In the estimation of physical activity, the subjects were asked how often they participated in brisk physical activity/exercise during their leisure-time. The term 'brisk' was defined as physical activity causing at least some sweating and getting out of breath, corresponding to moderate-to-vigorous intensity. Six response alternatives were 1) daily, 2) 4–6 times a week, 3) 2–3 times a week, 4) once a week, 5) 2–3 times a month, and 6) once a month or less often. Three relevant classes were formed by combining two consecutive categories (1+2, 3+4 and 5+6).