The present study was performed in accordance with the Helsinki Declaration of 1975, as revised in 2000, and the Institutional Review Board (IRB) of the University Hospital of the State University of Rio de Janeiro, Brazil approved of this study. Once considered eligible, all subjects read and signed an informed consent document that was approved by the IRB.

Study subjects presenting with type 1 diabetes (diagnosis based on a typical clinical presentation as well as the need to use insulin continuously since the diagnosis) of both sexes were recruited among patients who were followed up at a public hospital. Twenty-eight individuals with T1D diagnosed for more than 6 years, between 25 and 50 years of age, were recruited for the study. Twenty-two individuals (mean age 34 ± 7 years) completed the exercise protocol. There were 13 males (60 %) and 9 females (40 %) in the study group. The other six subjects (21 % of the subjects initially recruited for the study) left the study due to personal reasons.

The initial clinical evaluation included the patient’s history and physical examination as well as recording of the following anthropometric data: weight, height, waist circumference and body mass index (BMI). Blood pressure measurements were collected with patients in the supine position after 5 min in quiet surroundings; they were repeated twice with a two-minute interval between measurements. All measurements were performed before and after 12 weeks of physical training.

All patients included in the study were advised to maintain the same level of usual daily physical activity during the experimental protocol. Moreover, a regular diet for patients with type 1 diabetes was prescribed and supervised by the clinical staff of the tertiary care setting where they were recruited to certify that no significant changes occurred in the composition or total calories of the diet.

The diabetic patients included in the study were relatively young and did not present any co-morbidity. They were treated exclusively with insulin for diabetes control by an endocrinologist in the tertiary care setting, and none of them was taking other medications.

On the morning scheduled for the evaluation of cutaneous microcirculation, the patients presented in 12-h fasted condition for blood collection. Smokers should not have smoked or ingested caffeine from the night before until the completion of the tests. The following variables were measured: fasting and postprandial plasma glucose, total cholesterol, LDL and HDL cholesterol, triglycerides, transaminases, high-sensitivity C-reactive protein, gamma-glutamyl transferase, creatine kinase, urea, creatinine, albumin and uric acid by colorimetric reactions by using a Cobas Mira-machine (Roche). Blood samples were collected before and after the physical training period. LDL cholesterol was calculated using Friedewald’s formula. Serum samples were kept frozen at –80 °C until measurement of interleukin-6 (IL-6) levels with a commercial ELISA kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer’s instructions.

The study participants followed a non-supervised aerobic training protocol targeted at low intensity and corresponding to 40 % of their heart rate (HR) reserve (HRR). Resting HR was evaluated at the time of assessment of microvascular reactivity, and maximum HR was calculated using the formula [208 – (0.7 × age)] as described by Tanaka and colleagues [29]. HRR was calculated as proposed by Karvonen [30] [(HRmax – HRrest) x 40 % + HRrest] because it is highly correlated with VO₂ maximum [31]. The patients were advised to perform exercise sessions four times per week for 12 weeks that included alternating walking and running, in accordance with the patient’s fitness level, corresponding to 40 % of the HRR. Follow-up of the training period was conducted through weekly telephone contact. Heart rate was monitored using heart rate monitors (Polar Electro Oy, Kempele, Finland). During the first 4 weeks of training, 10 min were added per session every week to promote gradual progression in the range from 30 to 60 min during the remaining 8 weeks.

Microcirculatory tests were always performed after a 20-min period of rest in the supine position in a temperature-controlled room (23 ± 1 °C), thus excluding the influence of climate conditions from the results of the tests.

The capillary density, defined as the number of perfused capillaries per mm² of skin area, was assessed by high-resolution intra-vital color microscopy (Moritex, Cambridge, UK) using a video microscopy system with an epi-illuminated fiber optic microscope containing a 100-W mercury vapor lamp light source and an M200 objective with a final magnification of 200×. The dorsum of the non-dominant middle phalanx was used for image acquisition, which occurred while the patient sat comfortably in a constant temperature environment (23 ± 1 °C), as described in detail by Antonios et al. [32‐34]. Images were acquired and saved for posterior off-line analysis using a semi-automatic integrated system (Microvision Instruments, Evry, France). The mean capillary density was calculated as the arithmetic mean of the number of visible (i.e., spontaneously perfused) capillaries in three contiguous microscopic fields of 1 mm² each. A blood pressure cuff was then applied to the patient’s arm and inflated to suprasystolic pressure (50 mmHg above systolic arterial pressure) to completely interrupt blood flow for three minutes (testing post-occlusive reactive hyperemia, PORH). After cuff release, images were again acquired and recorded during the following 60–90 s, during which a maximal hyperemic response was expected to occur.

We assessed the variability of skin video capillaroscopy in a previous study [12]. The reproducibility of this methodology was assessed by examining an identical area of the finger skin; the intra-observer repeatability of data analysis was assessed by reading the same images in a blinded manner on two separate occasions (n = 20; coefficient of variability, 4.3 %). To assess inter-observer repeatability, a second observer independently assessed the capillary density in the same images (n = 15; coefficient of variability, 3.3 %).

Microvascular cutaneous reactivity was studied by laser Doppler flow monitoring (LDF), a method that has previously been standardized and validated [10, 35, 36]. Real-time variations in cutaneous microcirculatory flow were assessed through an LDF system (wavelength 780 nm; Periflux 5001, Perimed AB, Järfälla, Sweden) attached to a pharmacological micro-iontophoresis system (PeriIont, Perimed AB). The iontophoresis microelectrodes (PF 383, Perimed) were incorporated into the head of a laser probe (PF 481–1, Perimed), and the probe temperature was standardized to 32^°C to avoid variations in skin temperature and, consequently, in the measurements of microvascular flow. The drug-delivery electrodes were filled with 200 μl of 1 % ACh solution (Sigma Chemical Co., USA) and placed on the ventral surface of the forearm, away from visible subcutaneous veins and areas of skin pigmentation. Neutral electrodes for current dispersion were placed 15 cm above the infusion electrodes, and reference points were marked and annotated to ensure reproducibility during the second examination, which took place at the end of the intervention period. After measuring the baseline flow for 5 min, four equal cumulative doses of ACh (anodal current) were applied at a constant intensity of 0.1 mA for 10, 20, 40 and 80 s, with total charges of 1, 2, 4 and 8 millicoulombs, respectively, allowing for a 120-s interval between doses. Recording of the microvascular flow induced by ACh was conducted for 10 min as follows: 2 min for each dose and 2 min to allow the flow to reach a plateau after the last dose. Using a new delivery electrode applied to a different location on the same forearm, four doses of a solution of 1 % sodium nitroprusside (SNP; Sigma Chemical CO, USA) dissolved in distilled water were delivered using a cathodal current (same charges and intervals as for ACh).

The laser Doppler output, which is semiquantitative, is expressed in arbitrary perfusion units (PUs) of output voltage (1 PU = 10 mV) in accordance with general consensus (European Laser Doppler Users Groups, London, 1992). An area under the flow response to ACh curve could be defined using the PeriSoft for Windows 2.5 software (Perimed, Järfälla, Sweden); this area was quantitatively measured and expressed in PU/s.

After measuring the resting capillary flow for 5 min using another laser probe (PF 457, Perimed) that had been positioned at the start of the recording session, the PORH test was performed on the same forearm of pharmacological stimulation. Following release of the pressure, the maximum flow and area under the PORH curve were measured. The mean value of the resting flow was considered the basal flow value. When the microvascular flow returned to its basal value after the PORH test (typically 5–10 min), the maximal skin microvascular vasodilatation was investigated with prolonged (20 min) local heating of the laser probe to 42^°C. The baseline microvascular flux value was calculated as described above.

The values were expressed as the means ± standard error of the means for variables with a normal distribution and as median (percentiles 25^th–75^th) for variables with a non-parametric distribution according to results of the Shapiro-Wilk normality test. The data were analyzed by two-tailed paired t tests or the two-tailed Wilcoxon signed-rank test as appropriate. The null hypothesis was rejected at P < 0.05.

The mean capillary density after exercise training was significantly higher than before training (Fig. 1). A similar pattern was found during the PORH test; the capillary density was significantly higher after the training period compared with before the training period (Fig. 1). The increase in the capillary density measured in the basal state was from 119 ± 19 to 134 ± 25 capillaries/mm² (P = 0.0013) and after PORH from 121 ± 24 to 140 ± 26 capillaries/mm² (P < 0.0001). Moreover, there was improvement in the capillary recruitment induced by the PORH test after training; the capillary density after the PORH test increased from 134 ± 25 to 140 ± 26 capillaries/mm² (P = 0.0012).

The skin microvascular vasodilation responses induced by either endothelial-dependent (ACh) or endothelial-independent (SNP) vasoactive drugs were not different before and after exercise training (Fig. 2). The same pattern of responses was observed after physiological endothelial-dependent stimulation with the PORH test and local heating (Fig. 2). The individual values of microvascular parameters obtained with LDF are presented in Additional file 1.

The present study has limitations that may have influenced the findings. Such limitations include the fact that there was no control (sedentary) group of patients. Considering that physical exercise is a classical non-pharmacologic intervention in cardiovascular and metabolic diseases, we consider that it could be unethical to have a group of patients not participating in the exercise training program. On the other hand, we could have used a control (run in) period when the patients could have been evaluated after a period of inactivity. However, the main drawback of this type of experimental design is the resulting decrease of adherence to the study protocol, which is typical of patients with chronic diseases, and could have resulted in a greater dropout rate from the study.

Finally, it is also worth mentioning that the increases in systemic capillarity and improvement of capillary recruitment in patients with type 1 diabetes were obtained with an exercise training protocol that can be easily performed in the everyday life of patients with chronic diseases.