using small sample sizes (N,10) report no change in HRV after exercise training [12,13,14]. However, an observational study by Reland et al. has shown that postmenopausal women exhibiting high levels of physical activity also exhibit high levels of parasympathetic indices assessed by HRV [15]. Recently, we reported an improvement in HRV characteristics accompanying an 8-week exercise training intervention period [16]. Given the aforementioned observations, we hypothesized that HRV would exhibit a dose effect following 6-months of exercise training in postmenopausal women. To test our hypothesis, we examined the HRV response of women participating in the Dose-Response to Exercise in Women study (DREW) who participated in 6-months of strictly monitored, moderate intensity exercise training at approximately 50%, 100%, and 150% of the minimal NIH Panel’s recommendation for physical activity. Methods We have recently published a complete description of the DREW design, methods, and primary outcomes for our current cohort. These two papers detail how we derived the sample size for our main outcomes, the randomization sequence and allocation procedures, blinding techniques, recruitment and adverse events [9,17]. In brief, DREW is a randomized, dose-response, double- blind, exercise training trial that complies with Declaration of Helsinki and comprised of a non-exercise control group and three exercise training groups exercising at incremental doses (50%, 100%, and 150%) of the minimal NIH Panel’s recommendation for energy expenditure. The study was originally reviewed annually by The Cooper Institute and subsequently approved by the Pennington Biomedical Research Center IRB for the continued analysis and publication of pertinent research findings. Prior to participation, all participants signed a written informed consent document outlining the procedures involved in the DREW study. The primary outcomes for DREW study included peak aerobic power and resting blood pressure [9,17]. The data presented herein is the result of a secondary analysis examining the potential HRV response to exercise training. The supporting CONSORT checklist for this trial is available as Checklist S1, and the protocol is outlined in [17]. After an initial run-in period, we randomized 464 postmeno- pausal women (45–75 y) to 1 of 3 exercise training groups or a non-exercise control for a 6-month intervention period. Study participants were sedentary (exercising,than 20 minutes; ,3 d/ wk; ,8000 steps/d assessed over the course of 1 week), overweight 2 or obese (BMI 25.0 to 43.0 kg/m ), and had a systolic blood pressure of 120.0 to 159.9 mm Hg. As previously reported, there was no difference in this cohort at baseline or following our intervention for fasting measures of cholesterol, triglycerides, and glucose [9]. We excluded women who had a history of stroke, heart attack, or any serious medical condition that prevented participants from adhering to the protocol or exercising safely. Following baseline testing, inclusive of HRV (see below), participants were randomized into respective treatment groups. Fitness Testing We conducted our fitness testing using a Lode Excalibur Sport cycle ergometer (Groningen, Netherlands), an electronic, rate- independent ergometer. Participants cycled at 30 Watts (W) for 2 min, 50 W for 4 min, followed by increases of 20 W every 2 min until they could no longer maintain a pedal cadence of 50 rpm. Breath-by-breath respiratory gases were measured using a Parvomedics True Max 2400 Metabolic Measurement Cart. Volume and gas calibrations were conducted before each test. Exercise & HRV After Menopause Gas-exchange variables (VO2, CO2 production, ventilation, and respiratory exchange ratio [RER]) were averaged every 15 s. Heart rate was measured directly from the ECG monitoring system. Ratings of perceived exertion (RPE) were obtained using the 20-point Borg scale. Fitness was defined as the mean of two exercise test assessments at both baseline and 6 months. The reproducibility of the two fitness tests (VO2abs) was examined and characterized by an intraclass correlation of 0.88 at both baseline and follow-up testing. Daily Physical Activity and Exercise Training To assess potential changes in non-supervised physical activity, all randomized participants wore a step counter (Accusplit Eagle, Japan) to record their daily steps. Exercisers removed their step counter during supervised exercise sessions. Cardiovascular exercise training consisted of having women expend 4, 8, or 12 kcal/kg per week (KKW). During their exercise training sessions, women alternated exercise training using a recumbent cycle ergometer or treadmill. The target training intensity for each exercise training session was 50% of each woman’s VO2max for each exercise modality. We used a computer-controlled exercise training management that allowed us to input of relevant data points on each woman (week of exercise, KKW dose according to group assignment, their HR associated with 50% VO2max, training HR zone, body weight, and number of visits per week). The computer then provided us with the appropriate power output (PO) for the cycle ergometer, and the correct speed and grade for the treadmill. By knowing the exact PO for the cycle ergometer and the treadmill, the total kilocalories expended each minute and the time needed to reach the target energy expenditure for the exercise session or for the week can be calculated. The duration of each individual session depends on the number of visits required to reach the target KKW. To monitor the participant’s caloric expenditure over the course of 6 months, we created a weekly tracking report, which was used to track and make calculate caloric adjustments. The number of calories expended per session was adjusted each week, within the limits of the study design so that the total number of calories expended is equal to the total number prescribed per week for the 24-wk program. This report also averaged the number of visits per week so that we can determine whether the participants are exercising at least two, but no more than four, sessions each week. During exercise, HR is monitored every 3–6 min using a Polar HR monitor (Polar Vantage XL and Polar Vantage NV). The target HR was used to monitor each woman’s performance during each session to ensure that she was exercising at the proper intensity. Monitoring HR allowed us to control exercise intensity and document the specific amount of exercise done during each session. As women improved their fitness, they worked at gradually higher PO and spent less time to expend the required KKW. We contacted participants if they missed a scheduled session so that arrangements could be made to bring them back on schedule as soon as possible. As previously reported, the non-exercise training control group maintained their current level of activity during the trial period and showed no significant increase in physical activity [9].Those women in the exercise training groups adhered well to their prescribed exercise training and attended 92% of their exercise training sessions [9]. Heart Rate Variability We examined HRV between 06:30 a.m. and 11:00 a.m. for 25 minutes in a semi-dark room (22–23uC) following a 12-hour fast. Participants abstained from consuming caffeine-containing products and alcoholic beverages for 12 hours and heavy exercise PLoS ONE | www.plosone.org 2 June 2008 | Volume 3 | Issue 6 | e2288