Table 4.

Summary of the studies evaluating the impact of different modalities and intensities of exercising in healthy, DM1 or DM2

Reference	Design	Major outcomes
Healthy animal models
(Milne and Noble 2002)	Sprague-Dawley rats remained sedentary or underwent one of seven exercise intensities, for which treadmill running speed (15, 18, 21, 24, 27, 30, and 33 m/min) on a 2% incline during 60 min.	Cardiac HSP70 was significantly elevated only when animals were exercised at 24 m/min and beyond. Similarly, HSP70 was elevated in vastus red portion at running speeds above 24 m/min but did not increase in vastus white portion until 27 m/min. In contrast, HSP70 content was initially elevated in the soleus but subsequently declined at the highest running speeds. Exercise-induced elevation of HSP70 is intensity dependent.
(Antunes-Neto et al. 2006)	Wistar rats remained sedentary or ran in treadmill for 60 min, in a progressive protocol, with velocities varying between 10 and 30 m/min, considered an extenuous exercise.	There was a correspondence between erythrocyte CAT and GR activities and leukocyte HSP70 levels, principally 3 h after the acute exercise. The HSP70 concentration remained elevated 4 h after exhaustion. The increase in levels of TBARS occurred principally after two hours of exercise. Thus, tissue damage occurred before the expression of any antioxidant system markers.
(Melling et al. 2007)	Male Sprague-Dawley rats remained sedentary or aerobically trained (8 wk, low intensity), and were further submitted to an acute exercise (60 min at 30 m/min)	Trained animals (independent of acute exercise) increased the myocardial HSP70 content and protein kinase A activity, without differences between the exercised groups. However, acute exercise (independent of previous training) increased the HSP70 mRNA and a significant increase in HSF1-HSE DNA binding compared with control animals. Acute-trained animals presented higher ERK1/2 phosphorylation.
(Silver et al. 2012)	HSP70 mRNA was characterized via in situ hybridization experiments analyzing fast-muscle, white vastus: 1, 3, 10, and 24 h after a single bout of intense treadmill running (1 h, 30 m/min, 6% grade), or after a full-body heating for 15 min at 40.0 °C (HS-40 °C) or 42.0°C (HS-42 °C) core body temperature.	HSP70 mRNA signal was significantly greater 1 h post-exercise and continued to rise until 3 h post-exercise.
(Akin et al. 2017)	Female Sprague-Dawley ran 60 min a day at 30 m/min for 4 consecutive days. The exercise was performed at 25 °C (WE group) or 4 °C (CE).	Exercise increased body temperature only in the WE group. Adrenal Hsp72 and Hsp25 levels were significantly higher in the WE group compare to the other groups.
(Dos Santos et al. 2020)	Mice ran 40 min to a progressive speed, starting at 5 m/min, 10 m/min, 15 m/min and 17 m/min.	The Hsp70 released during exercise-induced delayed-onset muscle soreness (DOMS) activates the microglial TLR4/IL-6/TNF-α pathway in the spinal cord.
Obese and/or diabetic animal models
(Molanouri Shamsi et al. 2016)	Streptozotocin-induced type 1 diabetic or healthy rats, trained or sedentary. Trained animals performed 5-week ladder climbing resistance training, with a 48 h-interval between the sessions.	In diabetic rats, resistance training decreased inflammatory cytokines and HSP70 protein levels in fast skeletal muscle, increased adipose tissue inflammatory cytokines and HSP70, and preserved fast-twitch flexor hallucis longus (FHL) muscle mass.
(Tsuzuki et al. 2017)	Otsuka Long-Evans Tokushima Fatty rats remained sedentary (Sed), trained in a thermal-neutral environment (NTr: 25 °C), or trained in a cold environment (CTr: 4 °C). Training was conducted 5 days/week for 10 weeks.	Cold environment attenuated the exercise-induced increase of HSP72 levels in the gastrocnemius muscle and liver. It also blunted the improvement in whole-body insulin resistance and lipid metabolism in type 2 diabetic rats induced by physical training.
Clinical trial
(Shastry et al. 2002)	Trained (TR) and untrained (UT) subjects, ran on a treadmill for 1 h at 70% VO2.	Baseline HSP70 levels in TR subjects were lower than in UT subjects, while HSP90 levels were similar in TR and UT subjects. Exercise at an intensity that is within normal limits for a moderately trained individual did not induce HSP70 production in leucocytes 15 and 24 h after the session.
(Liu et al. 2004)	Well-trained rowers strength underwent a training program consisted of 3 weeks high-intensity training (HIT) and 3 weeks low intensity endurance training (ET), followed by 1 week of recovery each (R1 and R2, respectively).	HSP70 increased significantly at the end of HIT, decreased at the end of R1, and remained unchanged throughout ET and R2. HSP70 mRNA increased significantly after HIT and decreased gradually afterwards.
(Fehrenbach et al. 2005)	Athletes performed a continuous treadmill run of 60 min (CR) with an intensity of 75% VO2 max, a long treadmill run of 120 min (LR) with an intensity of 60% VO2 max, an extensive interval training (IT) program of 35 min, with 88% VO2 max), or a competitive marathon run (MA) within 260 ± 39 min, with an intensity of 65% VO2.	The concentration of sHSP72 in the athletes’ plasma was increased immediately (0 h) after exercise. The highest levels of sHSP72 were detected directly after the MA. If both runs were performed with similar intensity, the running time as twice as long in MA resulted in a more than 2.5-fold increase in sHSP72 levels compared with LR. Further, the exercise duration was kept constant while the exercise intensity was varied from 80% to 60% VO2 max. The higher intensity resulted in a significantly higher post-exercise leukocytosis. Likewise, a significantly greater increase of sHSP72 was found in the serum of the athletes who performed the exercise at an intensity of 80% VO2 max. Running exercise of different duration and intensity significantly increased the level of soluble HSP72 (sHSP72) in the plasma/serum of the athletes.
(Peake et al. 2005)	Subjects submitted to (1) level treadmill running at 60% VO2 max (moderate-intensity trial) for 60 min; (2) level treadmill running at 85% VO2 max (high-intensity trial) for 60 min; (3) downhill treadmill running (10% gradient) at 60% VO2 max (downhill running trial) for 45 min.	The plasma concentrations of IL-1ra, IL-12p40, MCP-1 and HSP70 increased significantly after all three trials.
(Pittaluga et al. 2006)	Young subjects with different training levels (professional athletes and non-agonists (NA)). Subjects were submitted to cycle ergometer exhaustive test, with different intensities.	Agonistic training led to an oxidative insult (high baseline values of oxidized glutathione (GSSG), micronuclei and hemolysis). On the contrary, NA with the lowest level of training frequency showed a well-balanced profile at rest, but they were more susceptible to exercise-induced variations (GSSG/GSH and diene increased values). In vitro heat shock of lymphocytes showed increased HSP70 baseline expression, especially in NA-Int (indicating ability to counteract stress).
(Hom et al. 2012)	Healthy males underwent 11 days of heat acclimation. Subjects walked for 90 min (50 ± 8% VO2max) on a treadmill (3.5 mph, 5% grade), in an environmental chamber (33 °C, 30–50% relative humidity). On days 1, 4, 7, 10, and 11, peripheral blood mononuclear cells were isolated from pre and post-exercise blood samples.	Heat acclimation was achieved in the absence of significant changes in intracellular HSP70 and percent of lymphocytes during acclimation. Furthermore, there was no increased cellular heat tolerance during secondary ex vivo heat shock of the lymphocytes acquired from subjects during acclimation.
(Periard et al. 2012)	Male subjects cycled to exhaustion at 60 and 75 % VO2 in hot conditions (40 °C, 50 % RH).	A significant increase and correlation was observed between eHSP72 and eHSP27 concentrations at exhaustion. Rectal temperature and VO2 max were significant predictor variables of eHSP72 expression. The exercise-induced eHSP72 release may be duration, intensity and exhaustion dependent.
(Periard et al. 2015)	Male subjects cycled to exhaustion at 60 and 75 % VO2 in hot conditions (40 °C, 50 % RH).	iHSP72 concentration increased similarly at exhaustion relative to pre-exercise and then increased further at 24 h. Exercise to exhaustion at high (75 %) and moderate (60 % of VO2 max) intensities in the heat induces a similar increase in iHSP72 concentration in monocytes.
(Tuttle et al. 2015)	Physically active but not heat-acclimated participants exercised at lactate threshold in either temperate (20 °C, 50% relative humidity; RH) or hot (30 °C, 50% RH) environmental conditions. Within each condition, participants completed a flat running (temperate flat or hot flat) and a downhill running (temperate downhill or hot downhill), separated by at least 7 days.	Downhill running and exercise in hot conditions induced the largest stimuli for leukocyte Hsp72 and Hsp90 mRNA, which increased immediately after exercise. Rectal temperature increased significantly during downhill running compared with flat running. Significant correlations were observed between Hsp72 mRNA and peak exercising rectal temperature. Exercising rectal temperature appeared to be the major physiological stimuli with increased metabolic strain during hot conditions and activation of the innate immune response potentially also contributing.
(Murase et al. 2016)	Healthy sedentary young males performed 59 min of cycling exercise at 75 % VO2 max. Samples were collected before (Pre), immediately after (Post), and at 1, 2, 3, and 4 h after completion of the exercise.	Exercise stress elevated salivary eHSP70 levels, with a peak in its secretion 4 h post-exercise. However, it partially downregulated oral immune function.
(Cuthbert et al. 2019)	Recreationally trained males cycled for 1 hour at 60% VO2 peak in 7 °C, 20 °C, and 33 °C with biopsies taken pre and 3 hours post-exercise.	HSP70 mRNA was higher 3 h post-exercise when compared to pre-exercise but was not significantly different between temperatures. Skeletal muscle mRNA of cold shock proteins decrease, while HSP70 mRNA increases in response to a low to moderate intensity aerobic exercise bout.
Clinical trials with obese and/or diabetic humans
(Tiss et al. 2014)	Lean and obese humans, both non-diabetic, undergoing 3-month moderate physical exercise. The exercise regimen involved a combination of moderate-intensity aerobic exercise and resistance training using a treadmill or stationary bicycle. Each exercise session included 10-min warm-up and cooldown steps at 50–60% max HR and 40 min of the prescribed exercise program at 65–80% max HR.	Obese subjects had increased expression of HSP60, HSC70, HSP72, HSP90 and GRP94 and lower expression of DNAJB3/HSP40 in the SAT and PBMC. Higher levels of HSP72 and GRP94 proteins correlated positively with the indices of obesity (body mass index and percent body fat). This expression pattern was concomitant with increased inflammatory response in the adipose tissue as monitored by increased levels of IL-6, TNF-α and RANTES. Physical exercise reduced the expression of various HSPs in obese to normal levels observed in lean subjects with a parallel decrease in the endogenous levels of IL-6, TNF-α, and RANTES.
(Khadir et al. 2018)	Obese human with and without diabetes undergoing 3-month, 3x/week, physical exercise or remaining sedentary. The exercise protocol involved a combination of moderate-intensity aerobic exercise and resistance training using a treadmill or stationary bicycle. Each exercise session included 10-min warm-up and cooldown steps at 50–60% max HR and 40 min of the prescribed exercise program at 65–80% max HR.	Compared with obese adults without diabetes, HSP60 mRNA and protein levels were decreased in subcutaneous adipose tissue (SAT) in diabetic obese together with increased inflammatory marker expression and glycemic levels but lower VO2 Max. More interestingly, a 3-month physical exercise decreased endogenous levels of IL-6 and TNF-α. It also reduced the expression of HSP60 in SAT along with decreased HSP72 expression in the diabetes group. Interestingly, the downregulation of HSP60 in adults with diabetes was concomitant with the increased expression of the tissue inflammatory cytokines produced by macrophage upon TLR or Th1 activation, IL-6 and TNF-a. HSP60 levels in blood serum were lower in the diabetes groups. Indeed, HSP60 levels were increased by exercise in the diabetes group together with an increase in HSP72 levels, whereas clear decreases in IL-6 and TNF-a levels were noted in this group. However, an opposite pattern was observed in the non-diabetes group for HSP60 and HSP72, in addition to a decrease in inflammatory marker levels.
(Brinkmann et al. 2019)	Overweight/ obese men with T2DM performed endurance training (3 times per week for 3 months, moderate intensity). The analysis were done at T1 (6 weeks pre-training), T2 (1 week pre-training) and T3 (3 to 4 days post-training).	HSP70 was upregulated in the muscle vastus lateralis following endurance training. There was a fiber type-specific distribution of HSP70 with increased protein contents in type I fibers. A significant change in the fiber type distribution with an increase in type I fibers and a decrease in type II fibers was observed post-training.